Nucleic acid encoding product that provides plants with fungal resistance and related methods

ABSTRACT

The invention relates to the field of plant diseases, in particular to oomycete infections such as late blight, a disease of major importance to production of Solanaceae such as potato and tomato cultivars. The invention provides a method for providing a plant or its progeny with resistance against an oomycete infection comprising providing said plant or part thereof with a gene or functional fragment thereof comprising a nucleic acid, said nucleic acid encoding a gene product that is capable of providing a member of the Solanaceae with resistance against an oomycete fungus.

[0001] Late blight, caused by the oomycete pathogen Phytophthora infestans is world-wide the most destructive disease for potato cultivation. The disease also threatens the tomato crop. The urgency of obtaining resistant cultivars has intensified as more virulent, crop-specialised and pesticide resistant strains of the pathogen are rapidly emerging.

[0002] A way to prevent crop failures or reduced yields is the application of fungicides that prevent or cure an infection by P. infestans. However, the application of crop protectants is widely considered to be a burden for the environment. Thus, in several Western countries, legislation is becoming more restrictive and partly prohibitive to the application of specific fungicides, making chemical control of the disease more difficult. An alternative approach is the use of cultivars that harbour partial or complete resistance to late blight. Two types of resistance to late blight have been described and used in potato breeding. One kind is conferred by a series of major, dominant genes that render the host incompatible with specific races of the pathogen (race specific resistance). Eleven such R genes (R1-R11) have been identified and are believed to have originated in the wild potato species Solanum demissum, which is native to Mexico, where the greatest genetic variation of the pathogen is found. Several of these R genes have been mapped on the genetic map of potato (reviewed in Gebhardt and Valkonen, 2001 Annu. Rev. Phytopathol. 39: 79-102). R1 and R2 are located on chromosomes 5 and 4, respectively. R3, R6 and R7 are located on chromosome 11. Unknown R genes conferring race specific resistance to late blight have also been described in S. tuberosum ssp. andigena and S. berthaultii (Ewing et al., 2000 Mol. Breeding 6: 25-36). Because of the high level of resistance and ease of transfer, many cultivars contain S. demissum derived resistance. Unfortunately, S. demissum derived race specific resistance, although nearly complete, is not durable. Once newly bred cultivars are grown on larger scale in commercial fields, new virulences emerge in P. infestans that render the pathogen able to overcome the introgressed resistance. The second type of resistance, termed field resistance and often quantitative in nature, is thought to be race non-specific and more durable. Field resistance to late blight can be found in several Mexican and Middle and South American Solanum species (Rossi et al., 1986 PNAS 95:9750-9754).

[0003] Diploid S. bulbocastanum from Mexico and Guatemala is one of the tuber bearing species that is known for its high levels of field resistance to late blight (Niederhauser and Mills, 1953 Phytopathology 43: 456-457). Despite differences in endosperm balance numbers, introgression of the S. bulbocastanum resistance trait has been successful. Ploidy manipulations and a series of tedious bridge crosses has resulted in S. bulbocastanum derived, P. infestans resistant germplasm (Hermsen and Ramanna, 1969 Euphytica 18:27-35; 1973 Euphytica 22:457-466; Ramanna and Hermsen, 1971 Euphytica 20:470-481; Hermsen and De Boer, 1971 Euphytica 20:171-180). However, almost 40 years after the first crosses and intense and continuous breeding efforts by potato breeders in the Netherlands with this germplasm, late blight resistant cultivars still remain to be introduced on the market. Successful production of somatic hybrids of S. bulbocastanum and S. tuberosum has also been reported (Thieme et al., 1997 Euphytica 97(2):189-200; Helgeson et al., 1998 Theor Appl. Genet 96:738-742). Some of these hybrids and backcrossed germplasm were found to be highly resistant to late blight, even under extreme disease pressure. Despite reports of suppression of recombination, resistance in the backcrossed material appeared to be on chromosome 8 within an approximately 6 cM interval between the RFLP markers CP53 and CT64 (Naess et al., 2000 Theor. Appl Genet 101:697-704). A CAPS marker derived from the tomato RFLP probe CT88 cosegregated with resistance. Suppression of recombination between the S. bulbocastanum and S. tuberosum chromosomes forms a potential obstacle for successful reconstitution of the recurrent cultivated potato germplasm to a level that could meet the standards for newly bred potato cultivars. Isolation of the genes that code for resistance found in S. bulbocastanum and subsequent transformation of existing cultivars with these genes, would be a much more straight forward and quicker approach when compared to introgression breeding.

[0004] The cloning and molecular characterisation of numerous plant R genes conferring disease resistance to bacteria, fungi, viruses, nematodes, and insects has identified several structural features characteristic to plant R genes (reviewed in Dangl and Jones, 2001 Nature 411, 826-833). The majority are members of tightly linked multigene families and all R genes characterised so far, with the exception of Pto, encode leucine-rich repeats (LRRs), structures shown to be involved in protein-protein interactions. LRR containing R genes can be divided into two classes based on the presence of a putative tripartite nucleotide-binding site (NBS). R genes of the NBS-LRR class comprise motifs that are shared with animal apoptosis regulatory proteins (van der Biezen et al., 1998 Curr. Biol. 8, 226-227; Aravind et al., 1999 Trends Biochem. Sci. 24, 47-53) and can be subdivided into two subgroups based on their N-terminal domain, which either exhibits sequence similarity to the Drosophila Toll protein and the mammalian interleukin-1 receptor domain (TIR-NBS-LRR), or contains a potential leucine zipper or coiled-coil domain (CC-NBS-LRR; Pan et al., 2000 Genetics. 155:309-22). LRR R genes without an NBS encode transmembrane proteins, whose extracellular N-terminal region is composed of LRRs (Jones et al., 1994 Adv. Bot. Res.24, 89-167). These genes can be divided into two subgroups based on the presence of a cytosolic serine/threonine kinase domain (Song et al., 1995 Science, 270, 1804-1806). Four R genes have currently been cloned from potato. All four, including the S. demissum derived R1 gene conferring race specific resistance to late blight, belong to the CC-NBS-LRR class of plant R genes (Bendahmane et al., 1999 Plant Cell 11, 781-791; Bendahmane et al., 2000 Plant J. 21, 73-81; van der Vossen et al., 2000 Plant Journal 23, 567-576; Ballvora et al., 2002 Plant Journal 30, 361-371).

[0005] The invention provides an isolated or recombinant nucleic acid comprising a nucleic acid coding for the amino acid sequence of FIG. 8 or a functional fragment or a homologue thereof. The protein coded by said amino acid has been detected as being member of a cluster of genes identifiable by phylogenetic tree analysis, which thus far consists of the proteins Rpi-blb, RGC1-blb, RGC3-blb and RGC4-blb (herein also called the Rpi-blb gene cluster) of FIG. 9.

[0006] Phylogenetic tree analysis is carried out as follows. First a multiple sequence alignment is made of the nucleic acid sequences and/or preferably of the deduced amino acid sequences of the genes to be analysed using CLUSTALW (http://www2.ebi.ac.uk/clustalw), which is in standard use in the art. ClustalW produces a .dnd file, which can be read by TREEVIEW (http://taxonomy.zoology.gla.ac.uk/rod/rod.html). The phylogenetic tree depicted in FIG. 9A is a phylogram.

[0007] Phylogenetic studies of the deduced amino acid sequences of Rpi-blb, RGC1-blb, RGC3-blb, RGC4-blb and those of the most similar genes from the art (as defined by the BLASTX) derived from diverse species, using the Neighbour-Joining method of Saitou and Nei (1987 Molecular Biology and Evolution 4, 406-425), shows that corresponding genes or functional fragments thereof of the Rpi-blb gene cluster can be placed in a separate branch (FIG. 9A).

[0008] Sequence comparisons between the four members of the Rpi-blb gene cluster identified on 8005-8 BAC clone SPB4 show that sequence homology within the Rpi-blb gene cluster varies between 70% and 81% at the amino acid sequence level. The deduced amino acid sequence of Rpi-blb shares the highest overall homology with RGC3-blb (81% amino-acid sequence identity; Table 4). When the different domains are compared it is clear that the effector domains present in the N-terminal halves of the proteins (coiled-coil and NBS-ARC domains) share a higher degree of homology (91% sequence identity) than the C-terminal halves of these proteins which are thought to contain the recognition domains (LRRs; 71% amino acid sequence identity). Comparison of all four amino-acid sequences revealed a total of 104 Rpi-blb specific amino acid residues (FIG. 10). The majority of these are located in the LRR region (80/104). Within the latter region, these specific residues are concentrated in the LRR subdomain xxLxLxxxx. The relative frequency of these specific amino-acid residues within this LRR subdomain is more than two times higher (28.3%) than that observed in the rest of the LRR domain (12.3%). The residues positioned around the two conserved leucine residues in the consensus xxLxxLxxxx are thought to be solvent exposed and are therefore likely to be involved in creating/maintaining recognition specificity of the resistance protein.

[0009] Sequences of additional members of the Rpi-blb gene cluster can be obtained by screening genomic DNA or insert libraries, e.g. BAC libraries with primers based on signature sequences of the Rpi-blb gene. Screening of various Solanum BAC libraries with primer sets A and/or B (Table 2 and FIG. 7) identified numerous Rpi-blb homologues derived from different Solanum species. Alignment of these additional sequences with those presented in FIG. 10 will help identify additional members of the Rpi-blb gene cluster and specific amino acid residues therein responsible for P. infestans resistance specificity. Furthermore, testing additional sequences in the above described phylogenetic tree analyses, e.g. using the Neighbour-Joining method of Saitou and Nei (1987 Molecular Biology and Evolution 4, 406-425), provides additional identification of genes belonging to the Rpi-blb gene cluster.

[0010] The invention provides the development of an intraspecific mapping population of S. bulbocastanum that segregated for race non-specific resistance to late blight. The resistance was mapped on chromosome 8, in a region located 0.3 cM distal from CT88. Due to the race non-specific nature of the resistance, S. bulbocastanum late blight resistance has always been thought to be R gene independent. However, with the current invention we demonstrate for the first time that S. bulbocastanum race non-specific resistance is in fact conferred by a gene bearing similarity to an R gene of the NBS-LRR type.

[0011] The invention further provides the molecular analysis of this genomic region and the isolation by map based cloning of a DNA-fragment of the resistant parent that harbours an R gene, designated Rpi-blb. This DNA-fragment was subcloned from an approximately 80 kb bacterial artificial chromosome (BAC) clone which contained four complete R gene-like sequences in a cluster-like arrangement. Transformation of a susceptible potato cultivar by Agrobacterium tumefaciens revealed that one of the four R gene-like sequences corresponds to Rpi-blb that provides the race non-specific resistance to late blight. Characterisation of the Rpi-blb gene showed that it is a member of the NBS-LRR class of plant R genes. The closest functionally characterised sequences of the prior art are members of the 12 resistance gene family in tomato. These sequences have an overall amino acid sequence identity of approximately 32% with that of Rpi-blb.

[0012] Thus, in a first embodiment, the invention provides an isolated or recombinant nucleic acid, said nucleic acid encoding a gene product having the sequence of Rpi-blb or a functional fragment thereof that is capable of providing a member of the Solanaceae family with race non-specific resistance against an oomycete pathogen.

[0013] Isolation of the gene as provided here that codes for the desired resistance trait against late blight and subsequent transformation of existing potato and tomato cultivars with this gene now provides a much more straightforward and quicker approach when compared to introgression breeding. The results provided here offer possibilities to further study the molecular basis of the plant pathogen interaction, the ecological role of R genes in a wild Mexican potato species and are useful for development of resistant potato or tomato cultivars by means of genetic modification.

[0014] In contrast to the R genes cloned and described so far, the gene we provide here is the first isolated R gene from a Solanum species that provides race non-specific resistance against an oomycete pathogen. Notably, the invention provides here a nucleic acid wherein said Solanum species that is provided with the desired resistance comprises S. tuberosum. In particular, it is the first gene that has been isolated from a phylogenetically distinct relative of cultivated potato, S. bulbocastanum, for which it was shown by complementation assays, that it is functional in S. tuberosum. These data imply that the gene Rpi-blb can easily be applied in potato production without a need for time-consuming and complex introgression breeding.

[0015] The following definitions are provided for terms used in the description and examples that follow.

[0016] Nucleic acid: a double or single stranded DNA or RNA molecule.

[0017] Oligonucleotide: a short single-stranded nucleic acid molecule.

[0018] Primer the term primer refers to an oligonucleotide that can prime the synthesis of nucleic acid.

[0019] Homology: homology is the term used for the similarity or identity of biological sequence information. Homology may be found at the nucleotide sequence and/or encoded amino acid sequence level. For calculation of precentage identity the BLAST algorithm can be used (Altschul et al., 1997 Nucl. Acids Res. 25:3389-3402) using default parameters or, alternatively, the GAP algorithm (Needleman and Wunsch, 1970 J. Mol. Biol. 48:443-453), using default parameters, which both are included in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., USA. BLAST searches assume that proteins can be modelled as random sequences. However, many real proteins comprise regions of nonrandom sequences which may be homopolymeric tracts, short-period repeats, or regions enriched in one or more amino acids. Such low-complexity regions may be aligned between unrelated proteins even though other regions of the protein are entirely dissimilar. A number of low-complexity filter programs can be employed to reduce such low-complexity alignments. For example, the SEG (Wooten and Federhen, 1993 Comput. Chem. 17:149-163) and XNU (Clayerie and States, 1993 Comput. Chem. 17:191-201) low-complexity filters can be employed alone or in combination. As used herein, ‘sequence identity’ or ‘identity’ in the context of two protein sequences (or nucleotide sequences) includes reference to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognised that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acids are substituted for other amino acid residues with similar chemical properties (e.g. charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percentage sequence identity may be adjusted upwards to correct for the conservative nature of the substitutions. Sequences, which differ by such conservative substitutions are said to have ‘sequence similarity’ or ‘similarity’. Means for making these adjustments are well known to persons skilled in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is give a score of zero, a conservative substitution is given a score between 0 and 1. The scoring of conservative substitutions is calculated, e.g. according to the algorithm of Meyers and Miller (Computer Applic. Biol. Sci. 4:11-17, 1988).

[0020] As used herein, ‘percentage of sequence identity’ means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the amino acid sequence or nucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical amino acid or nucleic acid base residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Preferably the amino acid sequence of the protein of the invention shares at least 82% or higher homology with the sequence as depicted in FIG. 8. As shown in Table 4, the closest functionally characterised sequence of the prior art (members of the 12 Fusarium resistance gene cluster in tomato) has a much lower level of amino acid sequence identity than this (32% with respect to that of Rpi-blb). Homology within the gene cluster of the present invention varies between 70% and 81% at the amino acid sequence level.

[0021] Homologous nucleic acid sequences are nucleic acid sequences coding for a homologous protein defined as above. One example of such a nucleic acid is the sequence as provided in FIG. 6A. However, there are many sequences which code for a protein which is 100% identical to the protein as depicted in FIG. 8. This is due to the ‘wobble’ in the nucleotide triplets, where more than one triplet can code for one and the same amino acid. Thus, even without having an effect on the amino acid sequence of the protein the nucleotide sequence coding for this protein can be varied substantially. It is acknowledged that nucleotide sequences coding for amino acid sequences that are not 100% identical to said protein can contain even more variations. Therefore, the percentage identity on nucleic acid sequence level can vary within wider limits, without departing from the invention.

[0022] Promoter. the term “promoter” is intended to mean a short DNA sequence to which RNA polymerase and/or other transcription initiation factors bind prior to transcription of the DNA to which the promoter is functionally connected, allowing transcription to take place. The promoter is usually situated upstream (5′) of the coding sequence. In its broader scope, the term “promoter” includes the RNA polymerase binding site as well as regulatory sequence elements located within several hundreds of base pairs, occasionally even further away, from the transcription start site. Such regulatory sequences are, e.g., sequences that are involved in the binding of protein factors that control the effectiveness of transcription initiation in response to physiological conditions. The promoter region should be functional in the host cell and preferably corresponds to the natural promoter region of the Rpi-blb resistance gene. However, any heterologous promoter region can be used as long as it is functional in the host cell where expression is desired. The heterologous promoter can be either constitutive or regulatable, tissue specific or not specific. A constitutive promoter such as the CaMV ³⁵S promoter or T-DNA promoters, all well known to those skilled in the art, is a promoter which is subjected to substantially no regulation such as induction or repression, but which allows for a steady and substantially unchanged transcription of the DNA sequence to which it is functionally bound in all active cells of the organism provided that other requirements for the transcription to take place is fulfilled. It is possible to use a tissue-specific promoter, which is driving expression in those parts of the plant which are prone to pathogen infection. In the case of Phytophthora a promoter which drives expression in the leaves, such as the ferredoxin promoter, can be used. A regulatable promoter is a promoter of which the function is regulated by one or more factors. These factors may either be such which by their presence ensure expression of the relevant DNA sequence or may, alternatively, be such which suppress the expression of the DNA sequence so that their absence causes the DNA sequence to be expressed. Thus, the promoter and optionally its associated regulatory sequence may be activated by the presence or absence of one or more factors to affect transcription of the DNA sequences of the genetic construct of the invention. Suitable promoter sequences and means for obtaining an increased transcription and expression are known to those skilled in the art.

[0023] Terminator: the transcription terminator serves to terminate the transcription of the DNA into RNA and is preferably selected from the group consisting of plant transcription terminator sequences, bacterial transcription terminator sequences and plant virus terminator sequences known to those skilled in the art.

[0024] Gene: the term “gene” is used to indicate a DNA sequence which is involved in producing a polypeptide chain and which includes regions preceding and following the coding region (5′-upstream and 3′-downstream sequences) as well as intervening sequences, the so-called introns, which are placed between individual coding segments (so-called exons) or in the 5′-upstream or 3′-downstream region. The 5′-upstream region may comprise a regulatory sequence that controls the expression of the gene, typically a promoter. The 3′-downstream region may comprise sequences which are involved in termination of transcription of the gene and optionally sequences responsible for polyadenylation of the transcript and the 3′ untranslated region. The term “resistance gene” is an isolated nucleic acid according to the invention said nucleic acid encoding a gene product that is capable of providing a plant with resistance against a pathogen, more specifically said plant being a member of the Solanaceae family, more preferably potato or tomato, said pathogen more specifically being an oomycete pathogen, more specifically Phytophthora, more specifically Phytophthora infestans, said nucleic acid preferably comprising a sequence as depicted in FIG. 8 or part thereof, or a homologous sequence with essentially similar functional and structural characteristics. A functionally equivalent fragment of such a resistance gene or nucleic acid as provided by the invention encodes a fragment of a polypeptide having an amino acid sequence as depicted in FIG. 8 or part thereof, or a homologous and/or functionally equivalent polypeptide, said fragment exhibiting the characteristic of providing at least partial resistance to an oomycete infection such as caused by P. infestans when incorporated and expressed in a plant or plant cell.

[0025] Resistance gene product: a polypeptide having an amino acid sequence as depicted in FIG. 8 or part thereof, or a homologous and/or functionally equivalent polypeptide exhibiting the characteristic of providing at least partial resistance to an oomycete infection such as caused by P. infestans when incorporated and expressed in a plant or plant cell.

[0026] Functionally equivalents of the protein of the invention are proteins that are homologous to and are obtained from the protein depicted in FIG. 8 by replacing, adding and/or deleting one or more amino acids, while still retaining their pathogen resistance activity. Such equivalents can readily be made by protein engineering in vivo, e.g. by changing the open reading frame capable of encoding the protein so that the amino acid sequence is thereby affected. As long as the changes in the amino acid sequences do not altogether abolish the activity of the protein such equivalents are embraced in the present invention. Further, it should be understood that equivalents should be derivable from the protein depicted in FIG. 8 while retaining biological activity, i.e. all, or a great part of the intermediates between the equivalent protein and the protein depicted in FIG. 8 should have pathogen resistance activity. A great part would mean 30% or more of the intermediates, preferably 40% or more, more preferably 50% or more, more preferably 60% or more, more preferably 70% or more, more preferably 80% or more, more preferably 90% or more, more preferably 95% or more, more preferably 99% or more.

[0027] Preferred equivalents are equivalents in which the leucine rich repeat region is highly homologous to the LRR region as depicted in FIG. 8. Other preferred equivalents are equivalents wherein the N-terminal effector domain is essential the same as the effector domain of Rpi-blb.

[0028] The protein of the invention comprises a distinct N-terminal effector domain and a leucine rich repeat domain. It is believed that conservation of these regions is essential for the function of the protein, although some variation is allowable. However, the other parts of the protein are less important for the function and may be more susceptible to change.

[0029] In order to provide a quick and simple test if the modified proteins and/or the gene constructs capable of expressing said modified proteins which are described here or any new constructs which are obvious to the person skilled in the art after reading this application indeed can yield a resistance response the person skilled in the art can perform a rapid transient expression test known under the name of ATTA (Agrobacterium tumefaciens Transient expression Assay). In this assay (of which a detailed description can be found in Van den Ackerveken, G., et al., Cell 87, 1307-1316, 1996) the nucleotide sequence coding for the modified protein which is to be tested is placed under control of the CaMV ³⁵S promoter and introduced into an Agrobacterium strain which is also used in protocols for stable transformation. After incubation of the bacteria with acetosyringon or any other phenolic compound which is known to enhance Agrobacterium T-DNA transfer, 1 ml of the Agrobacterium culture is infiltrated into an in situ plant leaf (from e.g. a tobacco or potato or tomato plant) by injection after which the plants are placed in a greenhouse and infected with a pathogen, preferably P. infestans. After 2-5 days the leaves can be scored for occurrence of resistance symptoms.

[0030] In the present invention we have identified and isolated the resistance gene Rpi-blb, which confers race non-specific resistance to Phytophthora infestans. The gene was cloned from a Solanum bulbocastanum genotype that is resistant to P. infestans. The isolated resistance gene according to the invention can be transferred to a susceptible host plant using Agrobacterium mediated transformation or any other known transformation method, and is involved in conferring the host plant resistant to plant pathogens, especially P. infestans. The host plant can be potato, tomato or any other plant, in particular a member of the Solanaceae family that may be infected by such a plant pathogen. The present invention provides also a nucleic acid sequence coding for this protein or a functional equivalent thereof, preferably comprising the Rpi-blb gene, which is depicted in FIG. 6.

[0031] With the Rpi-blb resistance protein or functionally equivalent fragment thereof according to the invention, one has an effective means of control against plant pathogens, since the gene coding for the protein can be used for transforming susceptible plant genotypes thereby producing genetically transformed plants having a reduced susceptibility or being preferably resistant to a plant pathogen. In particular, a plant genetically transformed with the Rpi-blb resistance gene according to the invention has a reduced susceptibility to P. infestans.

[0032] In a preferred embodiment the Rpi-blb resistance gene comprises the coding sequence provided in FIG. 6A or any homologous sequence or part thereof preceded by a promoter region and/or followed by a terminator region. The promoter region should be functional in plant cells, and preferably correspond to the native promoter region of the Rpi-blb gene. However, a heterologous promoter region that is functional in plant cells can be used in conjunction with the coding sequences.

[0033] In addition the invention relates to the Rpi-blb resistance protein which is encoded by the Rpi-blb gene according to the invention and which has an amino acid sequence provided in FIG. 8, or a functional equivalent thereof.

[0034] The signal that triggers the expression of the resistance gene in the wild-type S. bulbocastanum or in the transgenic plants of the invention is probably caused by the presence of a pathogen, more specifically the pathogen P. infestans. Such systems are known for other pathogen-plant interactions (Klement, Z., In: Phytopathogenic Prokaryotes, Vol. 2, eds.: Mount, M. S. and Lacy, G. H., New York, Academic Press, 1982, pp. 149-177), and use of this system can be made to increase the applicability of the resistance protein resulting in a resistance to more pathogens (see EP 474 857). This system makes use of the elicitor compound derived from the pathogen and the corresponding resistance gene, wherein the resistance gene when activated by the presence of the elicitor would lead to local cell death (hypersensitive reaction). In case of the present resistance gene, the corresponding elicitor component has not yet been disclosed, but it is believed that this is achievable by a person skilled in the art. Once the elicitor component is isolated it will be possible to transform the gene coding for said elicitor together with the gene coding for the resistance protein into plant, whereby one of the genes is under control of a pathogen-inducible promoter. These promoters are well known in the art (e.g. prp1, Fis1, Bet v 1, Vst1, gst1, and sesquiterpene cyclase, but any pathogen-inducible promoter which is switched on after pathogen infection can be used). If the transgenic plant contains such a system, then pathogen attack which is able to trigger the pathogen-inducible promoter will cause production of the component which is under control of said promoter, and this, in connection with the other component being expressed constitutively, will cause the resistance reaction to occur.

[0035] It will also be possible to mutate the resistance protein causing it to be in an active state (see EP1060257). Since this would permanently result in the resistance reaction to occur, which ultimately leads to local cell death, care should be taken not to constitutively express the resistance protein. This can be accomplished by placing the mutated resistance protein under control of a pathogen-inducible promoter, which not only would allow for expression of the active resistance protein only at times of pathogen attack, but would also allow a broader pathogen range to induce the hypersensitive reaction. Mutation of threonine and serine residues to aspartic acid and glutamic acid residues frequently leads to activation, as was shown in many proteins of which the activity is modulated by phosphorylation, e.g. in a MAPK-activated protein (Engel et al., 1995, J. Biol. Chem. 270, 27213-27221), and in a MAP-kinase-kinase protein (Huang et al.,1995 Mol. Biol.Cell 6, 237-245). Also C- and N-terminal as well as internal deletion mutants of these proteins can be tested for suitable mutants.

[0036] A more undirected way of identifying interesting mutants of which constitutive activity is induced is through propagation of the protein-encoding DNA in so-called E. coli ‘mutator’ strains.

[0037] A rapid way of testing all made mutants for their suitability to elicit a hypersensitive response is through a so-called ATTA assay (Van den Ackerveken, G., et al., Cell 87, 1307-1316, 1996). Many mutants can be screened with low effort to identify those that will elicit an HR upon expression.

[0038] The invention also provides a vector comprising a nucleic acid as provided herein, said nucleic acid encoding a gene product that is capable of providing a member of the Solanaceae family with resistance against an oomycete pathogen, or a functionally equivalent isolated or recombinant nucleic acid in particular wherein said member comprises S. tuberosum or Lycopersicon esculentum.

[0039] The invention also provides a host cell comprising a nucleic acid or a vector according to the invention. An example of said host cell is provided in the detailed description herein. In a particular embodiment, said host cell comprises a plant cell. As a plant cell a cell derived from a member of the Solanaceae family is preferred, in particular wherein said member comprises S. tuberosum or Lycopersicon esculentum. From such a cell, or protoplast, a transgenic plant, such as transgenic potato plant or tomato plant with resistance against an oomycete infection can arise. The invention thus also provides a plant, or tuber root, fruit or seed or part or progeny derived thereof comprising a cell according to the invention.

[0040] Furthermore, the invention provides a proteinaceous substance, exhibiting the characteristic of providing at least partial resistance to an oomycete infection such as caused by P. infestans when incorporated and expressed in a plant or plant cell. In particular such a proteinaceous substance is provided that is encoded by a nucleic acid according to the invention. In a preferred embodiment, the invention provides a proteinaceous substance comprising an amino acid sequence as depicted in FIG. 8 or a functional equivalent thereof. Preferably, such a functional equivalent will comprise one or more sequences which are relatively unique to Rpl-blb in comparison to RGC3-blb, RGC-blb and RGC4-blb. Such sequences can be spotted in the alignment (see FIG. 10A) and would be the sequences RPLLGEM, AKMEKEKLIS, KHSYTHMM, FFYTLPPLEKFI, GDSTFNK, NLYGSGMRS, LQYCTKLC, GSQSLTCM, NNFGPHI, TSLKIYGFRGIH, IIHECPFLTLS, RICYNKVA, and KYLTISRCN. It is believed that one or more of these sequences provide the functional characteristics of the protein Rpl-blb.

[0041] Furthermore, the invention provides a binding molecule directed at a nucleic acid according to the invention. For example, the Rpi-blb gene can be used for the design of oligonucleotides complementary to one strand of the DNA sequence as depicted in FIG. 7 and Table 2. Such oligonucleotides as provided herein are useful as probes for library screening, hybridisation probes for Southern/Northern analysis, primers for PCR, for use in a diagnostic kit for the detection of disease resistance and so on. Such oligonucleotides are useful fragments of an isolated or recombinant nucleic acid as provided herein, said nucleic acid encoding a gene product that is capable of providing a member of the Solanaceae family with resistance against an oomycete fungus, or a functionally equivalent isolated or recombinant nucleic acid, in particular wherein said member comprises S. tuberosum or Lycopersicon esculentum. They can be easily selected from a sequence as depicted in FIG. 6 or part thereof. A particular point of recognition comprises the LRR domain as identified herein. Such a binding molecule according to the invention is used as a probe or primer, for example provided with a label, in particular wherein said label comprises an excitable moiety which makes it useful to detect the presence of said binding molecule.

[0042] The invention furthermore provides a method for selecting a plant or plant material or progeny thereof for its susceptibility or resistance to an oomycete infection comprising testing at least part of said plant or plant material or progeny thereof for the presence or absence of a nucleic acid, said nucleic acid encoding a gene product that is capable of providing a member of the Solanaceae family with resistance against an oomycete fungus, or for the presence of said gene product, said method preferably comprising contacting at least part of said plant or plant material or progeny thereof with a binding molecule according the invention and determining the binding of said molecule to said part. Said method is particularly useful wherein said oomycete comprises P. infestans, allowing to select plants or planting material for resistance against late blight, for example wherein said plant or material comprises S. tuberosum. It is believed that by the phylogenetic tree analysis as discussed above, proteins that are highly homologous to Rpi-blb and which would yield resistance against plant pathogens could be easily idientified. An example for this is the detection of the three highly homologous proteins RGC1-blb, RGC3-blb and RGC4-blb, which have not yet been shown to yield resistance to P. infestans, but which are nevertheless believed to be involved in pathogen resistance in plants.

[0043] Also, the invention provides use of a nucleic acid or a vector or a cell or a substance or a binding molecule according to the invention in a method for providing a plant or its progeny with at least partial resistance against an oomycete infection, in particular wherein said oomycete comprises P. infestans especially wherein said plant comprises S. tuberosum, said method for providing a plant or its progeny with at least partial resistance against an oomycete infection comprising providing said plant or part thereof with a gene coding for a resistance protein or functional fragment thereof comprising a nucleic acid, said resistance protein being capable of providing a member of the Solanaceae family with resistance against an oomycete fungus, or providing said plant or part thereof with a nucleic acid or a vector or a cell or a substance according to the invention.

[0044] Furthermore, the invention provides an isolated S. bulbocastanum, or part thereof, such as a tuber or seed, susceptible to an oomycete infection caused by P. infestans.

[0045] The invention is further described in the detailed description below.

DESCRIPTION OF THE FIGURES

[0046]FIG. 1. Geographical map of Mexico indicating the origin of Solanum bulbocastanum accessions used to isolate the Rpi-blb gene. The letters a, b and c indicate the relative geographical origins of the used S. bulbocastanum accessions.

[0047]FIG. 2. Genetic linkage maps of the Rpi-blb locus on chromosome 8 of S. bulbocastanum. Horizontal lines indicate the relative positions of markers linked to late blight resistance. Distances between markers are indicated in centimorgans. A. Genetic position of the Rpi-blb locus relative to markers TG513, CT88 and CT64 (n=508 genotypes). B. High density genetic linkage map of the Rpi-blb locus (n=2109 genotypes).

[0048]FIG. 3. Physical map of the Rpi-blb locus. A. Genetic and physical map of the S. bulbocastanum genomic region containing Rpi-blb. Vertical arrows indicate the relative positions of markers linked to resistance. Numbers above the horizontal line indicate the number of recombinants identified between the flanking markers in 2109 progeny plants. Rectangles represent bacterial artificial chromosome (BAC) clones. B. Relative positions of candidate genes for late blight resistance on BAC SPB4. C. Schematic representation of the Rpi-blb gene structure. Horizontal lines indicate exons. Open boxes represent coding sequence. Lines angled downwards indicate the position of a 678-nucleotide long intron sequence.

[0049]FIG. 4. Southern blot analysis of the BAC contig spanning the Rpi-blb locus. Names above each lane represent the names of BAC clones. The names of the restriction enzymes used to digest the BAC DNA prior to Southern blotting are indicated.

[0050]FIG. 5. Detached leaf disease assays. A. Resistant (left), intermediate (centre) and susceptible (right) phenotypes found in the S. bulbocastanum mapping population B8 6 days post inoculation (d.p.i) with P. infestans sporangiospore droplets. B. Genetic complementation for late blight resistance in potato. Characteristic disease phenotypes of leaves derived from transgenic potato plants harbouring RGC1-blb, RGC2-blb, -blb or RGC4-blb 6 d.p.i. with P. infestans sporangiospore droplets. Genetic constructs harbouring the RGCs were transferred to the susceptible potato cultivar Impala through Agrobacterium mediated transformation. C. Genetic complementation for late blight resistance in tomato. Characteristic disease phenotype of a tomato leaf derived from transgenic tomat plants harbouring Rpi-blb 6 d.p.i. with P. infestans sporangiospore droplets (left panel). The genetic construct harbouring Rpi-blb was transferred to the susceptible tomato cultivar Moneymaker through Agrobacterium mediated transformation.

[0051]FIG. 6. Nucleic acid sequences of the Rpi-blb gene cluster members. A. Coding nucleic acid sequence of the Rpi-blb gene. B. Coding nucleic acid sequence of the Rpi-blb gene including the intron sequence (position 428-1106). C. Sequence of the 5.2 kb ScaI genomic DNA fragment of S. bulbocastanum BAC SPB4 present in pRGC2-blb, the genetic construct used for genetic complementation for late blight resistance. The genomic fragment harbours the Rpi-blb gene including natural regulatory elements necessary for correct expression of the gene. The initiation codon (ATG position 1191-1193) and the termination codon (TAA position4781-4783) are underlined. D. Coding nucleic acid sequence of RGC1-blb including the intron sequence (position 428-708). E. Coding nucleic acid sequence of RGC3-blb including the intron sequence (position 428-1458). F. Coding nucleic acid sequence of RGC4-blb including intron sequences (positions 434-510, 543-618 and 743-1365).

[0052]FIG. 7. Relative primer positions. The horizontal bar represents the coding sequence of the Rpi-blb gene. Numbers represent nucleotide positions. Horizontal arrows indicate relative primer positions and orientations. GSP1 and GSP2 represent nested gene specific primers used for 3′ RACE experiments. GSP3 and GSP4 represent nested gene specific primers used for 5′ RACE experiments. A(F), A(R), B(F) and B(R) are primers used to amplify Rpi-blb homologues. The position of the restriction site NsiI used to make domain swaps between Rpi-blb homologues is indicated.

[0053]FIG. 8. Deduced Rpi-blb protein sequence. The amino acid sequence deduced from the DNA sequence of Rpi-blb is divided into three domains (A-C), as described in Example 6. Hydrophobic residues in domain A that form the first and fourth residues of heptad repeats of potential coiled-coil domains are underlined. Conserved motifs in R proteins are written in lowercase and in italic in domain B. Residues matching the consensus of the cytoplasmic LRR are indicated in bold in domain C. Dots in the sequence have been introduced to align the sequence to the consensus LRR sequence of cytoplasmic LRRs.

[0054]FIG. 9. Phylogenetic tree analysis. A. Phylogenetic tree of state of the art sequences which share some degree of homology to the deduced amino acid sequence of Rpi-blb and its gene cluster members RGC1-blb, RGC3-blb and RGC4-blb. The tree was made according to the Neighbour-Joining method of Saitou and Nei (1987 Molecular Biology and Evolution 4, 406-425). An asterix indicates that the gene has been assigned a function. The Rpi-blb gene cluster is boxed. B. Phylogenetic tree of state of the art sequences which share some degree of homology to the deduced amino acid sequence of Rpi-blb. Included in this analysis are the Rpi-blb homologous sequences B149-blb, SH10-tub, SH20-tub and T118-tar, sequences identified through PCR amplification using Rpi-blb gene cluster specific primers. C. Relative positions of state of the art DNA sequences which show significant homology to parts of the Rpi-blb gene sequence. Horizontal lines represent the relative positions of the homologous sequences. The degree of homology is indicated to the right of each line. The length of the homologous sequence is indicated above each line.

[0055]FIG. 10. Alignment of the predicted Rpi-blb gene product to the predicted protein sequences of Rpi-blb homologues A. Alignment of the deduced protein products encoded by Rpi-blb, RGC1-blb, RGC3-blb and RGC4-blb. The complete amino acid sequence of Rpi-blb is shown and amino acid residues from RGC1-blb, RGC3-blb and RGC4-blb that differ from the corresponding residue in Rpi-blb. Dashes indicate gaps inserted to maintain optimal alignment. Amino acid residues that are specific for Rpi-blb, when compared to those at corresponding positions in RGC1-blb, RGC3-blb and RGC4-blb, are highlighted in bold. The regions of the LRRs that correspond to the consensus L . . . L . . . L . . . L . . . C/N/S . . . a . . . aP are underlined. Conserved motifs in the NBS domain are indicated in lowercase. B. Alignment of the deduced protein products encoded by Rpi-blb, RGC1-blb, RGC3-blb, RGC4-blb, B149-blb, SH10-tub, SH₂₀-tub and T118-tar.

[0056]FIG. 11. Schematic overview of domain swaps made between Rpi-blb and homologues RGC1-blb and RGC3-blb. The vertical dotted line indicates the position of the NsiI site used to make the swaps. R and S indicate whether transgenic plants harbouring specific chimeric constructs are resistant or susceptible to late blight infection, respectively.

EXPERIMENTAL PART

[0057] For the mapping of the Rpi-blb resistance gene an intraspecific mapping population of S. bulbocastanum was developed. A crucial step in this process was the identification of susceptible S. bulbocastanum genotypes. For this purpose several S. bulbocastanum accessions originating from different clusters/areas in Mexico were analysed for P. infestans resistance or susceptibility in a detached leaf assay (Table 1 and FIG. 1). The screened accessions BGRC 8008 and BGRC 7999 contained no susceptible genotypes. However in the accessions BGRC 8005, BGRC 8006 and BGRC 7997, susceptibility was found in 9%, 7% and 14% of the analysed seedlings, respectively. A P. infestans susceptible clone of accession BGRC 8006 was subsequently selected and crossed with a resistant clone of accession BGRC 8005. The resulting Fl population was used to map the Rpi-blb locus and is hereafter referred to as the B8 population.

[0058] Initial screening of 42 B8 genotypes for resistance to P. infestans in a detached leaf assay suggested that P. infestans resistance in S. bulbocastanum accession 8005 could be caused by a single dominant R gene, or a tightly linked gene cluster. Of the 42 genotypes tested, 22 scored resistant and 16 susceptible in a repeated experiment. Resistance phenotypes of the remaining 4 seedlings remained unclear. In order to determine the chromosome position of this S. bulbocastanum resistance, B8 genotypes with an undoubted phenotype were used for marker analysis. The chromosome 8 specific marker TG330 (Table 2) was found to be linked in repulsion phase with the resistant phenotype, as only one recombinant was obtained between this marker and resistance in 12 B8 genotypes. Furthermore, chromosome 8 marker CT88 (Table 2) was found to be completely linked in repulsion phase to resistance, indicating that the locus responsible for resistance, designated Rpi-blb, was located in this region of chromosome 8. For this reason, tomato chromosome 8 specific markers that map proximal and distal to CT88 (TG513 and CT64; Tanksley et al., 1992 Genetics 132: 1141-1160; Table 2) were developed into CAPS markers and tested in 512 B8 genotypes with known resistance phenotypes. A total of five CT64-CT88 recombinant genotypes and 41 CT88-TG513 recombinant genotypes were identified in this screen (FIG. 2A). The resistance locus Rpi-blb was mapped 1 recombination event distal to marker CT88 (FIG. 2A).

[0059] Fine mapping of the Rpi-blb locus was carried out with CAPS markers derived from left (L) and right (R) border sequences of BAC clones isolated from a BAC library prepared from the resistant S. bulbocastanum genotype BGRC 8005-8. The BAC library was initially screened with markers CT88 and CT64. BAC clones identified with these markers were used as seed BACs for a subsequent chromosome walk to the Rpi-blb locus. A total of 2109 B8 genotypes were screened for recombination between markers TG513 en CT64. All recombinant genotypes (219/2109) were subsequently screened with all available markers in the CT88-CT64 genetic interval. These data together with the disease resistance data of each recombinant, obtained through detached leaf assays, positioned the Rpi-blb locus between markers SPB33L and B149R, a 0.1 cM genetic interval (4/2109 recombinants) physically spanned by the overlapping BAC clones SPB4 and B49 (FIGS. 2b and 3). Within this interval resistance cosegregated with the BAC end marker SPB42L, the sequence of which was highly homologous to partial NBS fragments from tomato (e.g. Q 194, Q137, Q152, Q153;Pan et al., 2000 Genetics 155: 309-322). Southern analyses of BAC clones spanning the SP33L-B 149R interval using a ³²P-labeled PCR fragment of marker SPB42L as a probe revealed the presence of at least 4 copies of this R gene like sequence within the Rpi-blb interval (FIG. 4). Moreover, all of these copies were present on BAC SPB4. Sequencing and annotation of the complete insert of this BAC clone indeed identified four complete R gene candidates (RGC1-blb, RGC2-blb, RGC3-blb and RGC4-blb) of the NBS-LRR class of plant R genes. A PCR-marker that was located in-between RGC1-blb and RGC4-blb revealed recombination between P. infestans resistance and RGC4-blb, ruling out the possibility of RGC4-blb being Rpi-blb. Despite this finding, all four RGCs were selected for complementation analysis.

[0060] Genomic fragments of approximately 10 kb harbouring RGC1-blb, RGC2-blb, RGC3-blb or RGC4-blb were subcloned from BAC SPB4 into the binary plant transformation vector pBINPLUS (van Engelen et al., 1995 Trans. Res. 4, 288-290) and transferred to a susceptible potato cultivar using standard transformation methods. Primary transformants were tested for P. infestans resistance as described in Example 1. Only the genetic construct harbouring RGC2-blb was able to complement the susceptible phenotype; 86% of the primary transformants harbouring RGC2-blb were resistant (Table 3) whereas all RGC1-blb, RGC3-blb and RGC4-blb containing primary transformants were completely susceptible to P. infestans. The resistant RGC2-blb containing transformants showed similar resistance phenotypes as the S. bulbocastanum resistant parent (FIG. 5). RGC2-blb was therefore designated the Rpi-blb gene, the DNA sequence of which is provided in FIG. 6.

EXAMPLE 1 Disease Assay

[0061] The phenotype of S. bulbocastanum and transgenic S. tuberosum genotypes for resistance to P. infestans was determined by detached leaf assays. Leaves from plants grown for 6 to 12 weeks in the greenhouse were placed in pieces of water-saturated florists foam, approximately 35×4×4 cm, and put in a tray (40 cm width, 60 cm length and 6 cm height) with a perforated bottom. Each leaf was inoculated with two droplets or more (25 μl each) of sporangiospore solution on the abaxial side. Subsequently, the tray was placed in a plastic bag on top of a tray, in which a water-saturated filter paper was placed, and incubated in a climate room at 17° C. and a 16 h/8 h day/night photoperiod with fluorescent light (Philips TLD50W/84HF). After 6 days, the leaves were evaluated for the development of P. infestans disease symptoms. Plants with leaves that clearly showed sporulating lesions 6 days after inoculation were considered to have a susceptible phenotype whereas plants with leaves showing no visible symptoms or necrosis at the side of inoculation in the absence of clear sporulation were considered to be resistant. The assay was performed with P. infestans complex isolate 655-2A (race 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11), which was obtained from Plant Research International BV (Wageningen, The Netherlands).

EXAMPLE 2 Mapping of the Rpi-blb Resistance Locus

[0062] Plant Material

[0063] In order to produce an intraspecific mapping population that segregated for the P. infestans resistance gene present in S. bulbocastanum accession BGRC 8005 (CGN 17692, PI 275193), a susceptible S. bulbocastanum genotype was required. Several S. bulbocastanum accessions originating from different clusters/areas in Mexico were analysed for P. infestans resistance or susceptibility in a detached leaf assay (Table 1 and FIG. 1). In accession BGRC 8008 and BGRC 7999 no susceptibility was detected. In accession BGRC 8005, BGRC 8006 and BGRC 7997 susceptibility was only present in 9%, 7% and 14% of the analysed seedlings, respectively. Thus, only a few susceptible S. bulbocastanum genotypes were obtained.

[0064] The intraspecific mapping population of S. bulbocastanum (B8) was produced by crossing a P. infestans susceptible clone of accession BGRC 8006 with a resistant clone of accession BGRC 8005. DNA of 2109 progeny plants was extracted from young leaves according to Doyle and Doyle (1989 Focus 12, 13-15).

[0065] CAPS Marker Analysis

[0066] For PCR analysis, 15 μl reaction mixtures were prepared containing 0.5 μg DNA, 15 ng of each primer, 0.2 mM of each dNTP, 0.6 units Taq-polymerase (15 U/μl, SphaeroQ, Leiden, The Netherlands), 10 mM Tris-HCl pH 9, 1.5 mM MgCl₂, 50 mM KCl, 0.1% Triton X-100 and 0.01% (w/v) gelatin. The PCRs were performed using the following cycle profile: 25 seconds DNA denaturation at 94° C., 30 seconds annealing (see Table 1) and 40 seconds elongation at 72° C. As a first step in PCR-amplification DNA was denatured for 5 min at 94° C. and finalised by an extra 5 min elongation step at 72° C. The amplification reactions were performed in a Biometra® T-Gradient or Biometra® Uno-II thermocycler (Westburg, Leusden, The Netherlands). Depending on the marker, the PCR product was digested with an appropriate restriction enzyme. An overview of the markers including primer sequences, annealing temperature and restriction enzymes, is given in Table 2. Subsequently, the (digested) PCR products were analysed by electrophoresis in agarose or acrylamide gels. For acrylamide gel analysis, the CleanGel DNA Analysis Kit and DNA Silver Staining Kit (Amersham Pharmacia Biotech Benelux, Roosendaal, the Netherlands) were used.

[0067] Genetic Mapping of the Rpi-blb Locus

[0068] Initially a small group of 42 progeny plants of the B8 population was screened for resistance to P. infestans in a detached leaf assay. Plants with leaves that clearly showed sporulating lesions 6 days after inoculation were considered to have a susceptible phenotype whereas plants with leaves showing no visible symptoms or necrosis at the side of inoculation in the absence of clear sporulation were considered to be resistant. Of the 42 seedlings, 22 scored resistant and 16 susceptible. The phenotype of the remaining 4 seedlings remained unclear in this initial phase. These data indicated that resistance could be due to a single dominant gene or a tightly linked gene cluster. In order to determine the chromosome position, seedlings with a reliable phenotype were used for marker analysis. Chromosome 8 marker TG330 was found to be linked in repulsion with the resistant phenotype, as only one recombinant was obtained between this marker and resistance in 12 B8 seedlings. Furthermore, chromosome 8 marker CT88 was found to be completely linked in repulsion phase to resistance, indicating that a resistance gene was located on chromosome 8.

[0069] Subsequently, chromosome 8 specific markers that had been mapped proximal and distal to CT88 (Tanksley et al., 1992 Genetics 132: 1141-1160) were developed to CAPS markers. In order to map these markers more precisely, another 512 individuals of the B8 population were screened for late blight resistance using the detached leaf disease assay. Simultaneously, plants were scored for the markers CT64, CT88 and TG513. For 5 seedlings, recombination was detected between markers CT64 and CT88, while 41 seedlings were recombinant between markers CT88 and TG513 (FIG. 2A). The resistance gene Rpi-blb was mapped in between markers CT64 and CT88. In this stage, the positioning of CT88 proximal to Rpi-blb was based on only one recombined seedling.

[0070] In order to determine the position of Rpi-blb more precisely relative to the available markers, another 1555 seedlings of the B8 population were grown and analysed for recombination between the markers TG513 and CT64. Thus, a total of 2109 individual offspring clones of the B8 population were screened. Recombination between markers TG513 en CT64 was detected in 219 of these seedlings (10.4 cM). All of the recombinants were screened with marker CT88 and phenotyped for the resistance trait by making use of the detached leaf assay. In agreement with earlier results, the Rpi-blb gene was mapped in between markers CT88 and CT64 (FIG. 2B).

EXAMPLE 3 Construction of a S. bulbocastanum BAC Library and Construction of a Contiguous BAC Contig Spanning the Rpi-blb Locus

[0071] BAC Library Construction

[0072] A resistant clone of S. bulbocastanum (blb) accession BGRC 8005 (CGN 17692, PI 275193) heterozygous for the Rpi-blb locus, was used as source DNA for the construction of a genomic BAC library, hereafter referred to as the 8005-8 BAC library. High molecular weight DNA preparation and BAC library construction were carried out as described in Rouppe van der Voort et al. (1999 MPMI 12:197-206). Approximately 130.000 clones with an average insert size of 100 kb, which corresponds to 15 genome equivalents were finally obtained. A total of approximately 83.000 individual clones were stored in 216 384-well microtiter plates (Invitrogen, The Netherlands) containing LB freezing buffer (36 mM K₂HPO₄, 13.2 mM KH₂PO₄, 1.7 mM citrate, 0.4 mM MgSO₄, 6.8 mM (NH₄)₂SO₄, 4.4% V/V glycerol, 12.5 pg/ml chloramphenicol in LB medium) at −80° C. Another 50.000 clones were stored as bacterial pools containing ˜1000 white colonies. These were generated by scraping the colonies from the agar plates into LB medium containing 18% glycerol and 12.5 μg/ml chloramphenicol using a sterile glass spreader. These so-called super pools were also stored at −80° C.

[0073] Screening of the BAC Library and Construction of a Physical Map of the Rpi-blb Locus

[0074] The 8005-8 BAC library was initially screened with CAPS markers CT88 and CT64. This was carried out as follows. For the first part of the library of approximately 83.000 clones stored in 384 well microtiter plates, plasmid DNA was isolated using the standard alkaline lysis protocol (Sambrook et al., 1989 in Molecular cloning: a laboratory manual 2^(nd) edn, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.) from pooled bacteria of each plate to produce 216 plate pools. To identify individual BAC clones carrying the CAPS markers the plate pools were screened by PCR. Once an individual plate pool was identified as being positive for a particular CAPS marker the positive row and positive column were identified through a two dimensional PCR screening. For this purpose, the mother 384-well plate was replicated twice on LB medium containing chloramphenicol (12.5 pg/ml). After growing the colonies for 16 h at 37° C. one plate was used to scrape the 24 colonies of each row together and the other plate was used to scrape the 16 colonies of each column together. Bacteria of each row or column were resuspended in 200 μl TE buffer. CAPS marker analysis on 5 μl of these bacterial suspensions was subsequently carried out leading to the identification of single positive BAC clones. For the second part of the library, stored as 50 pools of approximately 1000 clones, plasmid DNA was isolated from each pool of clones using the standard alkaline lysis protocol and PCR was carried out to identify positive pools. Bacteria corresponding to positive pools were diluted and plated on LB agar plates containing chloramphenicol (12.5 μg/ml). Individual white colonies were subsequently picked into 384-well microtiter plates and single positive BAC clones subsequently identified as described above. Names of BAC clones isolated from the super pools carry the prefix SP (e.g. SPB33).

[0075] Insert sizes of BAC clones were estimated as follows. Positive BAC clones were analysed by isolating plasmid DNA from 2 ml overnight cultures (LB medium supplemented with 12.5 mg/ml chloramphenicol) using the standard alkaline lysis miniprep protocol and resuspended in 20 μl TE. Plasmid DNA (10 μl) was digested with 5 U NotI for 3 h at 37° C. to free the genomic DNA from the pBeloBAC11 vector. The digested DNA was separated by CHEF electrophoresis in a 1% agarose gel in 0.5×TBE at 4° C. using a BIORAD CHEF DR II system (Bio-Rad Laboratories, USA) at 150 volts with a constant pulse time of 14 sec for 16 h.

[0076] Screening of the 8005-8 BAC library with marker CT88 identified two positive BAC clones: B139 and B180, with potato DNA inserts of 130 and 120 kb, respectively (FIG. 3A). Digestion of the CT88 PCR product generated from these BAC clones and several resistant and susceptible progeny plants of the B8 mapping population with MboI revealed that BAC 139 carried the CT88 allele that was linked in cis to resistance. To identify the relative genome position of BAC B 139, pairs of PCR primers were designed based on the sequence of the right (R) and left (L) ends of the insert. BAC end sequencing was carried out as described in Example 4 using 0.5 μg of BAC DNA as template. Polymorphic CAPS markers were developed by digesting the PCR products of the two parent genotypes of the B8 population and of two resistant and two susceptible progeny genotypes with several 4-base cutting restriction enzymes (Table 2). Screening of the 37 CT88-CT64 recombinant B8 genotypes mapped 5 of the 7 CT88-Rpi-blb recombinants between CT88 and B139R, indicating that marker B 139R was relatively closer to the Rpi-blb locus than marker CT88. Screening of the 216 plate pools with B139R did not lead to the identification of a positive BAC clone. Screening of the 50 super pools identified the positive BAC clones SPB33 and SPB42 with DNA inserts of 85 and 75 kb, respectively (FIG. 3A). Screening of the complete BAC library with SPB33L identified the positive BAC clones B149 and SPB4. BAC clone SPB4 contained the SPB33L allele that was linked in cis to resistance whereas BAC clone B149 did not. However, screening of the CT88-CT64 recombinant panel with B 149R revealed that this BAC spanned the Rpi-blb locus. B 149R was separated from the Rpi-blb locus by two recombination events (FIG. 3A). Screening of the 8005-8 BAC library with B149R identified BAC clone B49 as having the B149R allele that was linked in cis to resistance. This BAC clone together with BAC clone SPB4 therefore formed a BAC contig that spanned the Rpi-blb locus (FIG. 3).

EXAMPLE 4 Sequence Analysis of BAC SPB4 and Identification of Resistance Gene Candidates within the Rpi-blb Locus

[0077] Within the SPB33L-B 149R interval resistance cosegregated with BAC end marker SPB42L, the sequence of which was highly homologous to partial NBS fragments from tomato (e.g. Q194, Q137, Q97, Q152, Q153; Pan et al., 2000 Genetics 155:309-22). Southern analyses of BAC clones spanning the SPB33L-B149R interval using a ³²P-labeled PCR fragment of marker SPB42L as a probe revealed the presence of at least 4 copies of this R gene like sequence within the Rpi-blb interval (FIG. 4). Moreover, all of these copies were present on BAC SPB4. The DNA sequence of BAC clone SPB4 was therefore determined by shotgun sequence analysis. A set of random subclones with an average insert size of 1.5 kb was generated. 10 pg of CsCl purified DNA was sheared for 6 seconds on ice at 6 amplitude microns in 200 μl TE using an MSE soniprep 150 sonicator. After ethanol precipitation and resuspension in 20 μl TE the ends of the DNA fragments were repaired by T4 DNA polymerase incubation at 11° C. for 25 minutes in a 50 μl reaction mixture comprising 1×T4 DNA polymerase buffer (New England BioLabs, USA), 1 mM DTT, 100 pM of all 4 dNTP's and 25 U T4 DNA polymerase (New England Biolabs, USA), followed by incubation at 65° C. for 15 minutes. The sheared DNA was subsequently separated by electrophoresis on 1% SeaPlaque LMP agarose gel (FMC). The fraction with a size of 1.5-2.5 kb was excised from the gel and dialysed against 50 ml TE for 2 hr at 4° C. Dialysed agarose slices were then transferred to a 1.5 ml Eppendorf tube, melted at 70° C. for 5 min, digested with 1 unit of GELASE (Epicentre Technologies, USA) per 100 mg of agarose gel for 1 hr at 45° C., and the DNA was subsequently precipitated. The 1.5-2.5 kb fragments were ligated at 16° C. in a EcoRV restricted and dephosphorylated pBluescript SK+vector (Stratagene Inc.). The ligation mixture was subsequently used to transform ElectroMAX E. coli DH10B competent cells (Life Technologies, UK) by electroporation using the BioRad Gene Pulser. Settings on the BioRad Gene Pulser were as recommended for E. coli by the manufacturer. The cells were spread on Luria broth (LB) agar plates containing ampicillin (100 μg/ml), 5-bromo-4-chloro-3-indolyl-β-D-galactoside (Xgal) (64 pg/ml) and isopropyl-1-thio-β-D-galactoside (IPTG) (32 μg/ml). Plates were incubated at 37° C. for 24 hours. Individual white colonies were grown in 96-well flat-bottom blocks (1.5 ml Terrific Broth medium containing 100 μg/ml ampicillin).

[0078] Plasmid DNA was isolated using the QIAprep 96 Turbo Miniprep system in conjunction with the BioRobot™ 9600 (QIAGEN) according to the manufacturers instructions. Sequencing reactions were performed using ABI PRISM BigDye™ Terminator cycle sequencing kit (Stratagene) according to the manufacturer's instructions. All clones were sequenced bi-directionally using universal primers. Sequence products were separated by capillary electrophoresis on a Perkin Elmer ABI 3700 DNA Analyzer. The automated assembly of the shotgun reads was carried out using the Phred-Phrap programs (Ewing and Green, 1998 Genome Research 8, 186-194; Ewing et al., 1998 Genome Research 8, 175-185). A total of 835 reads provided an overall BAC sequence coverage equal to 5×. Gaps between contigs were closed by primer walking or through a combinatorial PCR approach. The sequence was finally edited at Phred quality 40 (1 error every 10,000 nt) by manual inspection of the assembly using the Gap4 contig editor and re-sequencing of all low-quality regions. The complete sequence of the insert of BAC SPB4 consisted of 77,283 nucleotides.

[0079] Analysis of the contiguous sequence of BAC SPB4 using the computer programme GENSCAN (Burge and Karlin, 1997 J. Mol. Biol. 268, 78-94), GENEMARK (Lukashin and Borodovsky, 1998 NAR 26, 1107-1115) and BLASTX (Altschul et al., 1990 J. Mol. Biol. 215, 403-410) identified four complete R gene candidate sequences (RGC1-blb, RGC2-blb, RGC3-blb and RGC4-blb) belonging to the NBS-LRR class of plant R genes. A CAPS marker designed in between RGC1-blb and RGC4-blb, marker RGC1-4 revealed recombination between P. infestans resistance and RGC4-blb, ruling out the possibility of RGC4-blb being Rpi-blb (FIG. 3A and B). Despite this finding, all four RGCs were selected for complementation analysis.

EXAMPLE 5 Complementation Analysis

[0080] Subcloning of Candidate Genes and Transformation to Agrobacterium tumefaciens

[0081] Genomic fragments of approximately 10 kb harbouring RGC1-blb, RGC2-blb, RGC3-blb or RGC4-blb were subcloned from BAC clone SPB4 into the binary plant transformation vector pBINPLUS (van Engelen et al., 1995 Trans. Res. 4, 288-290). Restriction enzyme digestion of BAC clone SPB4 DNA and subsequent size selection was carried out as follows. Aliquots of ˜1 μg DNA were digested with 1U, 0.1U or 0.01U of Sau3AI restriction enzym for 30 min. The partially digested BAC DNA was subjected to CHEF electrophoresis at 4° C. in 0.5×TBE using a linear increasing pulse time of 1-10 sec and a field strength of 6 V/cm for 16 hr. After electrophoresis, the agarose gel was stained with ethidium bromide to locate the region of the gel containing DNA fragments of approximately 10 kb in size. This region was excised from the gel using a glass coverslip and dialysed against 50 ml TE for 2 hr at 4° C. Dialysed agarose slices were then transferred to a 1.5 ml Eppendorf tube, melted at 70° C. for 5 min and digested with 1 unit of GELASE (Epicentre Technologies, USA) per 100 mg of agarose gel for 1 hr at 45° C. Ligation of the size selected DNA to BamHI-digested and dephosphorylated pBINPLUS and subsequent transformation of ElectroMAX E. coli DH10B competent cells (Life Technologies, UK) with the ligated DNA was carried as described in Example 5, using the BioRad Gene Pulser for electroporation. The cells were spread on Luria broth (LB) agar plates containing kanamycin (50 μg/ml), Xgal (64 μg/ml) and IPTG (32 μg/ml). Plates were incubated at 37° C. for 24 hours. Individual white colonies were grown in 96-well plates (100 μl LB medium containing 50 pg/ml kanamycin). A total of 480 clones were PCR screened for the presence of RGCs using primers SPB42LF and SPB42LR or RGC4F and RGC4R (Table 2.). Positive clones were selected for plasmid isolation and further characterisation. Identification of clones harbouring RGC1-blb, RGC2-blb, RGC3-blb or RGC4-blb was carried out by sequencing the SPB42L PCR fragments derived from positive clones. The relative position of the RGCs within a subclone was determined by sequencing the ends of the clone and subsequent comparison of the sequences to the complete BAC insert sequence. Finally four binary plasmids, pRGC1-blb, pRGC2-blb, pRGC3-blb and pRGC4-blb were selected and transferred to Agrobacterium tumefaciens strains AGLO (Lazo et al., 1991 Bio/Technology 9, 963-967), LBA4404 (Hoekema et al., 1983 Nature 303: 179-180) or UIA143 (Farrand et al.,1989 J. of Bacteriology 171, 5314-5321) either by electroporation using the BioRad Gene Pulser or by conjugation. Settings on the BioRad Gene Pulser were as recommended for A. tumefaciens by the manufacturer. Conjugation was carried out as described by Simon et al. (1983 Bio/Tech. 1, 784-791). The cells were spread on Luria broth (LB) agar plates containing kanamycin (100 mg/l) and rifampicin (50 mg/l). Plates were incubated at 28° C. for 48 hours. Small-scale cultures from selected colonies were grown in LB medium containing kanamycin (100 mg/l) and rifampicin (50 mg/l). Plasmid DNA was isolated as described previously and the integrity of the plasmids was verified by restriction analysis upon reisolation from A. tumefaciens and subsequent transformation to E. coli. A tumefaciens cultures harbouring a plasmid with the correct DNA pattern were used to transform a susceptible potato genotype.

[0082] Transformation of Susceptible Potato Cultivar

[0083]A. tumefaciens strains were grown for 2 days at 28° C. in 20 ml LB medium supplemented with 50 mg/l rifampicine and 25 mg/l kanamycin. Subsequently, 0.2 ml of A. tumefaciens culture was diluted in 10 ml LB medium containing the same antibiotics and grown overnight (28° C.). The overnight culture was centrifuged (30 min, 2647×g) and the pellet was resuspended in 50 ml MS medium (Murashige and Skoog, 1962 Physiol. Plant. 15, 473-497) supplemented with 30 g/l sucrose (MS30).

[0084] Certified seed potatoes of cultivar Impala were peeled and surface sterilised for 30 min. in a 1% sodium hypochlorate solution containing 0.1% Tween-20. Tubers were then washed thoroughly in large volumes of sterile distilled water (4 times, 10 min). Discs of approximately 2 mm thickness and 7 mm in diameter, were sliced from cylinders of tuber tissue prepared with a corkborer. The tuber discs were transferred into liquid MS30 medium containing A. tumefaciens and incubated for 15 min. After removing the A. tumefaciens solution, the tuber discs were transferred to regeneration medium containing MS30, 0.9 mg/l IAA, 3.6 mg/l zeatine riboside and 8 g/l agar (Hoekema et al., 1989 Bio/Technology 7, 273-278). The plates were incubated at 24° C., 16 hour day-length (Philips TLD50W/84HF). After 48 hours of co-cultivation, the tuber discs were rinsed for 5 min in liquid MS medium including antibiotics, 200 mg/l vancomycin, 250 mg/l cefotaxim and 75 mg/l kanamycin, and transferred to regeneration medium supplemented with the same antibiotics. The plates were incubated at 24° C., 16 hour day-length (Philips TLD50W/84HF). Every three weeks, the tuber discs were transferred to fresh medium. Regenerating shoots were transferred to MS30 medium containing 75 mg/l kanamycin. Rooting shoots were propagated in vitro and tested for absence of A. tumefaciens cells by incubating a piece of stem in 3 ml LB medium (3 weeks, 37° C., 400 rpm). One plant of each transformed regenerant was transferred to the greenhouse.

[0085] Complementation of the Susceptible Phenotype in Potato

[0086] Primary transformants were tested for P. infestans resistance as described in Example 1. Only the genetic construct harbouring RGC2-blb was able to complement the susceptible phenotype; 15 out of 18 RGC2-blb containing primary transformants were resistant (Table 3) whereas all RGC1-blb, RGC3-blb and RGC4-blb containing primary transformants were completely susceptible to P. infestans. The resistant RGC2-blb transformants showed similar resistance phenotypes as the S. bulbocastanum resistant parent (FIG. 5). RGC2-blb was therefore designated the Rpi-blb gene, the DNA sequence of which is provided in FIG. 6.

[0087] Transformation of Susceptible Tomato

[0088] Seeds of the susceptible tomato line Moneymaker were rinsed in 70% ethanol to dissolve the seed coat and washed with sterile water. Subsequently, the seeds were surface-sterilised in 1.5% sodium hypochlorite for 15 minutes, rinsed three times in sterile water and placed in containers containing 140 ml MS medium pH 6.0 (Murashige and Skoog, 1962 Physiol. Plant. 15, 473-497) supplemented with 10 g/l sucrose (MS10) and 160 ml vermiculite. The seeds were left to germinate for 8 days at 25° C. and 0.5 W/M2 light. Eight day old cotyledon explants were pre-cultured for 24 hours in Petri dishes containing a two week old feeder layer of tobacco suspension cells plated on co-cultivation medium (MS30 pH 5.8 supplemented with Nitsch vitamines (Duchefa Biochemie BV, Haarlem, The Netherlands), 0.5 g/l MES buffer and 8 g/l Daichin agar).

[0089] Overnight cultures of A. tumefaciens were centrifuged and the pellet was resuspended in cell suspension medium (MS30 pH 5.8 supplemented with Nitsch vitamines, 0.5 g/l MES buffer, pH 5.8) containing 200 pM acetosyringone to a final O.D.₆₀₀ of 0.25. The explants were then infected with the diluted overnight culture of A. tumefaciens strain UIA143 (Farrand et al., 1989 J. of Bacteriology 171, 5314-5321) containing the helper plasmid pCH32 (Hamilton et al., 1996 PNAS 93, 9975-9979) and pRGC2-blb for 25 minutes, blotted dry on sterile filter paper and co-cultured for 48 hours on the original feeder layer plates. Culture conditions were as described above.

[0090] Following the co-cultivation, the cotyledons explants were transferred to Petri dishes with selective shoot inducing medium (MS pH 5.8 supplemented with 10 g/l glucose, including Nitsch vitamines, 0.5 g/l MES buffer, 5 g/l agargel, 2 mg/l zeatine riboside, 400 mg/l carbenicilline, 100 mg/l kanamycine, 0.1 mg/l IAA) and cultured at 25° C. with 3-5 W/m² light. The explants were sub-cultured every 3 weeks onto fresh medium. Emerging shoots were dissected from the underlying callus and transferred to containers with selective root inducing medium (MS 10 pH 5.8 supplemented with Nitsch vitamines, 0.5 g/l MES buffer, 5 g/l agargel, 0.25 mg/l IBA, 200 mg/l carbenicillin and 100 mg/l kanamycine).

[0091] Complementation of the Susceptible Phenotype in Tomato

[0092] To investigate whether Rpi-blb could complement the susceptible phenotype in tomato, primary transformants of Moneymaker harbouring the Rpi-blb gene construct were initially challenged with the potato derived P. infestans isolates IP0655-2A and IP0428. Seven out of nine primary transformants were resistant (Table 3). In view of the observation that the tested potato P. infestans isolates were less virulent on tomato than on potato, the primary transformants were also tested with a P. infestans isolate collected from susceptible home garden tomato plants. Even though this isolate was significantly more virulent on Moneymaker than the previously tested ones, all 7 primary transformants remained resistant. These results illustrate the potential effectiveness of the Rpi-blb gene not only against complex isolates derived from potato but also to those specialised on tomato.

[0093] Molecular Analysis of Primary Transformants

[0094] RT-PCR Analysis

[0095] In order to produce cDNA, a mix of 19 μl containing 1 μg of total or polyA RNA, 0.25 mM of each dNTP, 50 mM Tris-HCl pH 8.3, 75 mM KCl, 3 mM MgCl₂, 10 mM DTT and 530 ng oligo d(T) primer, GCTGTCAACGATACGCTACGTAACGGCATGACAGTG(T)₁₈ was denatured (1 min 83° C.). Subsequently, the mix was placed at 42° C. and 1 μl reverse transcriptase (M-MLV reverse transcriptase, Promega Benelux b.v., Leiden, The Netherlands) was added. After 60 min, the mix was heated for 1 min at 99° C. and transferred to ice. 2 μl cDNA was used for standard PCR.

[0096] Rapid Amplification of cDNA Ends

[0097] The 5′ and 3′ ends of the Rpi-blb cDNA were determined by rapid amplification of cDNA ends (RACE) using the GeneRacer™ kit (Invitrogen™, The Netherlands). 3′ RACE was carried out with the primers GSP1 (5′-GAGGAATCCATCTCCCAGAG) and GSP2 (5′-GTGCTTGAAGAGATGATAATTCACGAG) in combination with the GeneRacer™ 3′ primer and GeneRacer™ 3′ nested primer. 5′ RACE was carried out on cDNA synthesised with the primer GSP3 (5′-GTCCATCTCACCAAGTAGTGG) using primers GSP4 (5′-GAAATGCTCAGTAACTCTCTGG) and GSP5 (5′-GGAGGACTGAAAGGTGTTGG) in combination with the GeneRacer™ 5′ primer and GeneRacer™ 5′ nested primer (FIG. 7).

EXAMPLE 6 Structure of the Rpi-blb Gene and the Corresponding Protein.

[0098] The size and structure of the Rpi-blb gene was determined by comparing the genomic sequence derived from the insert of pRGC2-blb with cDNA fragments generated by 5′ and 3′ rapid amplification of cDNA ends. RACE identified 5′ and 3′ Rpi-blb specific cDNA fragments of a single species, respectively, suggesting that the genomic clone encodes a single Rpi-blb specific transcript. The coding sequence of the Rpi-blb transcript is 2913 nucleotides The putative Rpi-blb transcript is estimated to be 3138 nucleotides (nt) and contains a 44 and 181 nt long 5′- and 3′-untranslated region (UTR), respectively. The Rpi-blb gene contains a single intron of 678 nt starting 428 nt after the translational ATG start codon of the gene (FIG. 3C).

[0099] The deduced open reading frame of the Rpi-blb gene encodes a predicted polypeptide of 970 amino acids with an estimated molecular weight of 110.3 kD (FIG. 8). Several functional motifs present in R genes of the NBS-LRR class of plant R genes are apparent in the encoded protein which can be subdivided into 3 domains (A, B and C; FIG. 8). The N-terminal part of the protein contains potential coiled-coil domains, heptad repeats in which the first and fourth residues are generally hydrophobic (domain A). Domain B harbours the NBS and other motifs that constitute the NB-ARC domain (ARC for Apaf-1, R protein, and CED-4) of R proteins and cell death regulators in animals (van der Biezen and Jones, 1998). This domain includes the Ap-ATPase motifs present in proteins of eukaryotic and prokaryotic origin (Aravind et al., 1999 Trends Biochem. Sci. 24, 47-53). The C-terminal half of Rpi-blb comprises a series of 19-20 irregular LRRs (domain C). The LRRs can be aligned according to the consensus sequence LxxLxxLxLxxC/N/SxxLxxLPxxa, where x designates any residue and “a” designates the positions of aliphatic amino acids, followed by a region of varying length. This repeat format approximates the consensus for cytoplasmic LRRs (Jones and Jones, 1997 Adv. Bot. Res.24, 89-167).

EXAMPLE 7 Natural Homologues and Artificial Variants of the Rpi-blb Gene

[0100] Natural Homologues

[0101] BLASTN homology searches with the coding DNA sequence of the Rpi-blb gene identified a number of sequences with significant homology to short stretches of the Rpi-blb gene (FIG. 9C). Nucleotides 549-1245 of the coding sequence of the Rpi-blb gene share 81-90% sequence identity to partial NBS fragments from tomato (e.g. Q194, Q137, Q198 and Q199; Pan et al., 2000 Genetics. 155:309-22). These homologous sequences vary in length between 525 and 708 nucleotides and are PCR fragments which were identified by systematically scanning the tomato genome using (degenerate) primer pairs based on ubiquitous NBS motifs (Pan et al., 2000 Genetics. 155:309-22; Leister et al., 1996 Nat Genet. 14:421-429). Another region of the Rpi-blb gene which shares significant homology to a state of the art sequence comprises nucleotides 76-805 of the coding sequence. This 729 nt long sequence shares 91% sequence identity to an EST from potato (EMBL database accession no. BG890602; FIG. 9C). The Rpi-blb gene sequence downstream of nucleotide 1245, comprising the LRR region, shares no significant homology to any state of the art sequence.

[0102] BLASTX homology searches with the coding sequence of the Rpi-blb gene revealed that amino acid sequence homology with various state of the art genes does not exceed 36% sequence identity (Table 4). The best BLASTX score was obtained with an NBS-LRR gene derived from Oryza sativa (36.5% amino acid sequence identity). NBS-LRR genes sharing an overall sequence homology of 27-36% amino-acid sequence identity with Rpi-blb can be found among others in Arabidopsis thaliana, Phaseolus vulgaris, Lycopersicon esculentum (Fusarium 12 gene cluster; Ori et al., 1997 Plant Cell, 9, 521-532; Simons et al, 1998 Plant Cell 10, 1055-1068), Zea mays, Hordeum vulgare and Lactuca sativa. Phylogenetic studies of the deduced amino acid sequences of Rpi-blb, RGCI-blb, RGC3-blb, RGC4-blb and those of the homologous state of the art genes (as defined by BLASTX) derived from diverse species, using the Neighbour-Joining method of Saitou and Nei (1987 Molecular Biology and Evolution 4, 406-425), shows that members of the Rpi-blb gene cluster can be placed in a separate branch (FIG. 9).

[0103] Sequence comparisons of the four RGCs of the Rpi-blb gene cluster identified on 8005-8 BAC clone SPB4 show that sequence homology within the Rpi-blb gene cluster varies between 70% and 81% at the amino acid level. The deduced amino acid sequence of Rpi-blb shares the highest overall homology with RGC3-blb (81% amino acid sequence identity; Table 4). When the different domains are compared it is clear that the N-terminal halves of the proteins (coiled-coil and NB-ARC domains) share a higher degree of homology (91% amino acid sequence identity) than the C-terminal halves of these proteins (LRRs; 71% amino acid sequence identity). The N-terminus of NBS-LRR proteins influences the requirement for downstream signalling components and is therefore thought to be the putative effector domain (Feys and Parker, 2000 Trends Genet 16:449-55). The C-terminal LRR region is implicated, by genetic studies, in elicitor recognition specificity (Ellis et al., 2000 Trends Plant Sci. 5:373-379; Dodds et al., 2001 Plant Cell 13:163-78).

[0104] Comparison of all four amino acid sequences revealed a total of 104 Rpi-blb specific amino acid residues (FIG. 10A). The majority of these are located in the LRR region (80/104). Within the latter region, these specific residues are concentrated in the LRR subdomain xxLxLxxxx. The relative frequency of these specific amino-acid residues within this LRR subdomain is more than two times higher (28.3%) than that observed in the rest of the LRR domain (12.3%). The residues positioned around the two conserved leucine residues in the consensus xxLxxLxxxx are thought to be solvent exposed and are therefore likely to be involved in creating/maintaining recognition specificity of the resistance protein.

[0105] Sequences of additional homologues of the Rpi-blb gene can be obtained by screening genomic DNA or insert libraries, e.g. BAC libraries with primers based on signature sequences of the Rpi-blb gene. Screening of various Solanum BAC libraries with primer sets A and/or B (Table 2 and FIG. 7) identified other Rpi-blb homologues derived from Solanum bulbocastanum (B149-blb), S. tuberosum (SH 10-tub and SH₂₀-tub) and S. tarijense (T118-tar). Comparison of all 8 protein sequences reduces the number of Rpi-blb specific amino acid residues to 51 (51/970; 5.25%) (FIG. 10B). The majority of these are located in the LRR region (42/51; 82%). The relative frequency of these specific amino-acid residues within the LRR subdomain xxLxlxxxx is 3.3 times higher than that observed in the rest of the LRR domain (18.8% versus 5.7%, respectively). These data clearly suggest that evolution of P. infestans resistance specificity within the Rpi-blb gene cluster has mainly evolved through shifts in Rpi-blb LRR specific residues.

[0106] Inclusion of the additional Rpi-blb homologues in the above described phylogenetic tree analyses, using the Neighbour-Joining method of Saitou and Nei (1987 Molecular Biology and Evolution 4, 406-425), further justifies phylogenetic tree analysis as a method to define Rpi-blb homologous sequences (FIG. 9B). Any functional R gene product which shares at least 70% sequence identity at the amino acid level will end up in the same branch as gene products of the the Rpi-blb gene cluster and can thus be defined as being a homologue of Rpi-blb.

[0107] Artificial Variants

[0108] Domain swaps between the different homologues can be made to ascertain the role of the different sequences in P. infestans resistance. The restriction enzyme NsiI for example, which recognises the DNA sequence ATGCAT present in the conserved MHD motif can be used to swap the complete LRR domain of Rpi-blb with that of RGC1-blb or RGC3-blb using techniques known to those skilled in the art. Chimeric variants of the Rpi-blb gene were made which encode the N-terminal half of Rpi-blb and the C-terminal half of RGC 1-blb or RGC3-blb and visa versa, i.e., the N-terminal half of RGCl-blb or RGC3-blb and the C-terminal half of Rpi-blb (FIG. 11). These variants were transformed to the susceptible potato genotype Impala and tested for P. infestans resistance. Chimeric RGC3-blb genes containing the LRR domain of Rpi-blb were resistant to P. infestans indicating that the specificity of the Rpi-blb gene is encoded by this part of the gene. TABLE 1 Overview of P. infestans susceptibility in different S. bulbocastanum accessions S. bulbocastanum accession # # # % CGN BGRC PI Plants R V susceptibility Cluster^(a) 17692 8005 275193 11 10 1 9 A 8006 275194 16 15 1 6 A 17693 8008 275198 19 18 0 B 17687 7997 243505 35 25 4 14 B 17688 7999 255518 19 19 0 0 C

[0109] TABLE 2 Overview of markers used for mapping Rpi-blb Restriction Marker Ori^(a) Sequence^(b) Annealing Temp (° C.) enzyme^(c) TG513 F CGTAAACGCACCAAAAGCAG 58 a.s. R GATTCAAGCCAGGAACCGAG TG330 F CAGCTGCCACAGCTCAAGC 56 TaqI R TACCTACATGTACAGTACTGC CT88 F GGCAGAAGAGCTAGGAAGAG 57 MboI R ATGGCGTGATACAATCCGAG F TTCAAGAGCTTGAAGACATAACA 60 a.s. R ATGGCGTGATACAATCCGAG CT64 F ACTAGAGGATAGATTCTTGG 56 CfoI R CTGGATGCCTTTCTCTATGT B139R F GATCAGAAGTGCCTTGAACC 56 TaqI R CAAGGAGCTTGGTCAGCAG SPB33L F ATTGCACAGGAGCAGATCTG 59 Hinfl R TGTAAGAGAGCAAGAGGCAC SPB42L F AGAGCAGTCTTGAAGGTTGG 58 CfoI R GATGGTAACTAAGCCTCAGG B149R F GACAGATTTCTCATAAACCTGC 58 MseI/XbaI R AATCGTGCATCACTAGAGCG RGC1-4 F TGTGGAGTAAGAGAGGAAGG 62 SspI/MseI R TCAGCTGAGCAGTGTGTGG A F ATGGCTGAAGCTTCATTCAAGTTCTG 60 R TCACACCGCTTGATCAGTTGTGGAC B F TRCATGAYCTMATCCATGATTTGC 60 R GMAATTTTGTGCCAGTCTTCTCC

[0110] TABLE 3 Complementation of late blight susceptibility in potato and tomato RGA-containing R plants/ plants/ RGA-containing Genotype^(a) transformants plants IMP(RGC1- 15/17^(b)  0/15 blb) 8/9^(d) 0/8 IMP(RGC2-  6/31^(c) 6/6 blb) 12/14^(d)  9/12 IMP(RGC3- 0/6^(c) — blb) 5/5^(d) 0/5 IMP(RGC4- 18/19^(b)  0/18 blb)  1/12^(c) 0/1 IMP(vector) 8/8^(b) 0/8  9/10^(d) 0/9 MM(RGC2-  9/11^(d) 7/9 blb)

[0111] TABLE 4 Comparison of nucleotide and amino acid sequence homology 8005-8 BAC SPB4 RGC3- RGC1- RGC4- Rice Arabidopsis Tomato blb blb blb RGC RGC I2C-1 Rpi-blb nt^(a) 88 84 81 — — — aa^(a) 81 76 70 36 32 32 N^(b) C^(b) N C N C 91 71 79 72 75 66

[0112]

1 63 1 9 PRT Artificial Sequence Description of Artificial Sequence concentration in LRR subdomain 1 Xaa Xaa Leu Xaa Leu Xaa Xaa Xaa Xaa 1 5 2 10 PRT Artificial Sequence Description of Artificial Sequence consensus 2 Xaa Xaa Leu Xaa Xaa Leu Xaa Xaa Xaa Xaa 1 5 10 3 7 PRT Artificial Sequence Description of Artificial Sequence sequence which is relatively unique to Rpi-blb protein 3 Arg Pro Leu Leu Gly Glu Met 1 5 4 10 PRT Artificial Sequence Description of Artificial Sequence sequence which is relatively unique to Rpi-blb protein 4 Ala Lys Met Glu Lys Glu Lys Leu Ile Ser 1 5 10 5 8 PRT Artificial Sequence Description of Artificial Sequence sequence which is relatively unique to Rpi-blb protein 5 Lys His Ser Tyr Thr His Met Met 1 5 6 12 PRT Artificial Sequence Description of Artificial Sequence sequence which is relatively unique to Rpi-blb protein 6 Phe Phe Tyr Thr Leu Pro Pro Leu Glu Lys Phe Ile 1 5 10 7 7 PRT Artificial Sequence Description of Artificial Sequence sequence which is relatively unique to Rpi-blb protein 7 Gly Asp Ser Thr Phe Asn Lys 1 5 8 9 PRT Artificial Sequence Description of Artificial Sequence sequence which is relatively unique to Rpi-blb protein 8 Asn Leu Tyr Gly Ser Gly Met Arg Ser 1 5 9 8 PRT Artificial Sequence Description of Artificial Sequence sequence which is relatively unique to Rpi-blb protein 9 Leu Gln Tyr Cys Thr Lys Leu Cys 1 5 10 8 PRT Artificial Sequence Description of Artificial Sequence sequence which is relatively unique to Rpi-blb protein 10 Gly Ser Gln Ser Leu Thr Cys Met 1 5 11 7 PRT Artificial Sequence Description of Artificial Sequence sequence which is relatively unique to Rpi-blb protein 11 Asn Asn Phe Gly Pro His Ile 1 5 12 12 PRT Artificial Sequence Description of Artificial Sequence sequence which is relatively unique to Rpi-blb protein 12 Thr Ser Leu Lys Ile Tyr Gly Phe Arg Gly Ile His 1 5 10 13 11 PRT Artificial Sequence Description of Artificial Sequence sequence which is relatively unique to Rpi-blb protein 13 Ile Ile His Glu Cys Pro Phe Leu Thr Leu Ser 1 5 10 14 8 PRT Artificial Sequence Description of Artificial Sequence sequence which is relatively unique to Rpi-blb protein 14 Arg Ile Cys Tyr Asn Lys Val Ala 1 5 15 9 PRT Artificial Sequence Description of Artificial Sequence sequence which is relatively unique to Rpi-blb protein 15 Lys Tyr Leu Thr Ile Ser Arg Cys Asn 1 5 16 54 DNA Artificial Sequence Description of Artificial Sequence oligo d(T) primer 16 gctgtcaacg atacgctacg taacggcatg acagtgtttt tttttttttt tttt 54 17 20 DNA Artificial Sequence Description of Artificial Sequence primer GSP1 17 gaggaatcca tctcccagag 20 18 27 DNA Artificial Sequence Description of Artificial Sequence primer GSP2 18 gtgcttgaag agatgataat tcacgag 27 19 21 DNA Artificial Sequence Description of Artificial Sequence primer GSP3 19 gtccatctca ccaagtagtg g 21 20 22 DNA Artificial Sequence Description of Artificial Sequence primer GSP4 20 gaaatgctca gtaactctct gg 22 21 20 DNA Artificial Sequence Description of Artificial Sequence primer GSP5 21 ggaggactga aaggtgttgg 20 22 22 PRT Artificial Sequence Description of Artificial Sequence consensus 22 Leu Xaa Xaa Leu Xaa Xaa Leu Xaa Leu Xaa Xaa Xaa Xaa Xaa Leu Xaa 1 5 10 15 Xaa Leu Pro Xaa Xaa Xaa 20 23 6 DNA Artificial Sequence Description of Artificial Sequence NsiI-site 23 atgcat 6 24 20 DNA Artificial Sequence Description of Artificial Sequence forward primer 24 cgtaaacgca ccaaaagcag 20 25 20 DNA Artificial Sequence Description of Artificial Sequence reverse primer 25 gattcaagcc aggaaccgag 20 26 19 DNA Artificial Sequence Description of Artificial Sequence forward primer 26 cagctgccac agctcaagc 19 27 21 DNA Artificial Sequence Description of Artificial Sequence reverse primer 27 tacctacatg tacagtactg c 21 28 20 DNA Artificial Sequence Description of Artificial Sequence forward primer 28 ggcagaagag ctaggaagag 20 29 20 DNA Artificial Sequence Description of Artificial Sequence reverse primer 29 atggcgtgat acaatccgag 20 30 23 DNA Artificial Sequence Description of Artificial Sequence forward primer 30 ttcaagagct tgaagacata aca 23 31 20 DNA Artificial Sequence Description of Artificial Sequence reverse primer 31 atggcgtgat acaatccgag 20 32 20 DNA Artificial Sequence Description of Artificial Sequence forward primer 32 actagaggat agattcttgg 20 33 20 DNA Artificial Sequence Description of Artificial Sequence reverse primer 33 ctggatgcct ttctctatgt 20 34 20 DNA Artificial Sequence Description of Artificial Sequence forward primer 34 gatcagaagt gccttgaacc 20 35 19 DNA Artificial Sequence Description of Artificial Sequence reverse primer 35 caaggagctt ggtcagcag 19 36 20 DNA Artificial Sequence Description of Artificial Sequence forward primer 36 attgcacagg agcagatctg 20 37 20 DNA Artificial Sequence Description of Artificial Sequence reverse primer 37 tgtaagagag caagaggcac 20 38 20 DNA Artificial Sequence Description of Artificial Sequence forward primer 38 agagcagtct tgaaggttgg 20 39 20 DNA Artificial Sequence Description of Artificial Sequence reverse primer 39 gatggtaact aagcctcagg 20 40 22 DNA Artificial Sequence Description of Artificial Sequence forward primer 40 gacagatttc tcataaacct gc 22 41 20 DNA Artificial Sequence Description of Artificial Sequence reverse primer 41 aatcgtgcat cactagagcg 20 42 20 DNA Artificial Sequence Description of Artificial Sequence forward primer 42 tgtggagtaa gagaggaagg 20 43 19 DNA Artificial Sequence Description of Artificial Sequence reverse primer 43 tcagctgagc agtgtgtgg 19 44 27 DNA Artificial Sequence Description of Artificial Sequence forward primer 44 atggctgaag ctttcattca agttctg 27 45 25 DNA Artificial Sequence Description of Artificial Sequence reverse primer 45 tcacaccgct tgatcagttg tggac 25 46 24 DNA Artificial Sequence Description of Artificial Sequence forward primer 46 trcatgayct matccatgat ttgc 24 47 23 DNA Artificial Sequence Description of Artificial Sequence reverse primer 47 gmaattttgt gccagtcttc tcc 23 48 2913 DNA Solanum bulbocastanum misc_feature (1)..(2913) /note=“Rpi-blb” 48 atggctgaag ctttcattca agttctgcta gacaatctca cttctttcct caaaggggaa 60 cttgtattgc ttttcggttt tcaagatgag ttccaaaggc tttcaagcat gttttctaca 120 attcaagccg tccttgaaga tgctcaggag aagcaactca acaacaagcc tctagaaaat 180 tggttgcaaa aactcaatgc tgctacatat gaagtcgatg acatcttgga tgaatataaa 240 accaaggcca caagattctc ccagtctgaa tatggccgtt atcatccaaa ggttatccct 300 ttccgtcaca aggtcgggaa aaggatggac caagtgatga aaaaactaaa ggcaattgct 360 gaggaaagaa agaattttca tttgcacgaa aaaattgtag agagacaagc tgttagacgg 420 gaaacaggtt ctgtattaac cgaaccgcag gtttatggaa gagacaaaga gaaagatgag 480 atagtgaaaa tcctaataaa caatgttagt gatgcccaac acctttcagt cctcccaata 540 cttggtatgg ggggattagg aaaaacgact cttgcccaaa tggtcttcaa tgaccagaga 600 gttactgagc atttccattc caaaatatgg atttgtgtct cggaagattt tgatgagaag 660 aggttaataa aggcaattgt agaatctatt gaaggaaggc cactacttgg tgagatggac 720 ttggctccac ttcaaaagaa gcttcaggag ttgctgaatg gaaaaagata cttgcttgtc 780 ttagatgatg tttggaatga agatcaacag aagtgggcta atttaagagc agtcttgaag 840 gttggagcaa gtggtgcttc tgttctaacc actactcgtc ttgaaaaggt tggatcaatt 900 atgggaacat tgcaaccata tgaactgtca aatctgtctc aagaagattg ttggttgttg 960 ttcatgcaac gtgcatttgg acaccaagaa gaaataaatc caaaccttgt ggcaatcgga 1020 aaggagattg tgaaaaaaag tggtggtgtg cctctagcag ccaaaactct tggaggtatt 1080 ttgtgcttca agagagaaga aagagcatgg gaacatgtga gagacagtcc gatttggaat 1140 ttgcctcaag atgaaagttc tattctgcct gccctgaggc ttagttacca tcaacttcca 1200 cttgatttga aacaatgctt tgcgtattgt gcggtgttcc caaaggatgc caaaatggaa 1260 aaagaaaagc taatctctct ctggatggcg catggttttc ttttatcaaa aggaaacatg 1320 gagctagagg atgtgggcga tgaagtatgg aaagaattat acttgaggtc ttttttccaa 1380 gagattgaag ttaaagatgg taaaacttat ttcaagatgc atgatctcat ccatgatttg 1440 gcaacatctc tgttttcagc aaacacatca agcagcaata tccgtgaaat aaataaacac 1500 agttacacac atatgatgtc cattggtttc gccgaagtgg tgttttttta cactcttccc 1560 cccttggaaa agtttatctc gttaagagtg cttaatctag gtgattcgac atttaataag 1620 ttaccatctt ccattggaga tctagtacat ttaagatact tgaacctgta tggcagtggc 1680 atgcgtagtc ttccaaagca gttatgcaag cttcaaaatc tgcaaactct tgatctacaa 1740 tattgcacca agctttgttg tttgccaaaa gaaacaagta aacttggtag tctccgaaat 1800 cttttacttg atggtagcca gtcattgact tgtatgccac caaggatagg atcattgaca 1860 tgccttaaga ctctaggtca atttgttgtt ggaaggaaga aaggttatca acttggtgaa 1920 ctaggaaacc taaatctcta tggctcaatt aaaatctcgc atcttgagag agtgaagaat 1980 gataaggacg caaaagaagc caatttatct gcaaaaggga atctgcattc tttaagcatg 2040 agttggaata actttggacc acatatatat gaatcagaag aagttaaagt gcttgaagcc 2100 ctcaaaccac actccaatct gacttcttta aaaatctatg gcttcagagg aatccatctc 2160 ccagagtgga tgaatcactc agtattgaaa aatattgtct ctattctaat tagcaacttc 2220 agaaactgct catgcttacc accctttggt gatctgcctt gtctagaaag tctagagtta 2280 cactgggggt ctgcggatgt ggagtatgtt gaagaagtgg atattgatgt tcattctgga 2340 ttccccacaa gaataaggtt tccatccttg aggaaacttg atatatggga ctttggtagt 2400 ctgaaaggat tgctgaaaaa ggaaggagaa gagcaattcc ctgtgcttga agagatgata 2460 attcacgagt gcccttttct gaccctttct tctaatctta gggctcttac ttccctcaga 2520 atttgctata ataaagtagc tacttcattc ccagaagaga tgttcaaaaa ccttgcaaat 2580 ctcaaatact tgacaatctc tcggtgcaat aatctcaaag agctgcctac cagcttggct 2640 agtctgaatg ctttgaaaag tctaaaaatt caattgtgtt gcgcactaga gagtctccct 2700 gaggaagggc tggaaggttt atcttcactc acagagttat ttgttgaaca ctgtaacatg 2760 ctaaaatgtt taccagaggg attgcagcac ctaacaaccc tcacaagttt aaaaattcgg 2820 ggatgtccac aactgatcaa gcggtgtgag aagggaatag gagaagactg gcacaaaatt 2880 tctcacattc ctaatgtgaa tatatatatt taa 2913 49 3592 DNA Solanum bulbocastanum misc_feature (1)..(3591) /note=“Rpi-blb including intron sequence (position 428-1106)” 49 atggctgaag ctttcattca agttctgcta gacaatctca cttctttcct caaaggggaa 60 cttgtattgc ttttcggttt tcaagatgag ttccaaaggc tttcaagcat gttttctaca 120 attcaagccg tccttgaaga tgctcaggag aagcaactca acaacaagcc tctagaaaat 180 tggttgcaaa aactcaatgc tgctacatat gaagtcgatg acatcttgga tgaatataaa 240 accaaggcca caagattctc ccagtctgaa tatggccgtt atcatccaaa ggttatccct 300 ttccgtcaca aggtcgggaa aaggatggac caagtgatga aaaaactaaa ggcaattgct 360 gaggaaagaa agaattttca tttgcacgaa aaaattgtag agagacaagc tgttagacgg 420 gaaacaggta ctcatcttaa attagtatta caacaactaa gtttatattc atttttttgg 480 caattatcaa attcagaaaa gggttaaata tactcatgtc ctatcgtaaa tagtgtatat 540 atacctctcg ttgtactttc gatctgaata tacttgtcaa atctggcaag ctcagaatca 600 aattatccac cccaactttt aaatactcga tatctttaga aatccacctg tctaactcat 660 ccactaccca ttccctttgc tttgaattct tttctttacc tataaacttg gaacactcga 720 tccgttttgc ttttcttaac aaagcagctc agagaaaaga ggttttcttc tattctgttt 780 ctctgtgtgc tgcacttggg tccttaatcc cattaaaaac agggcatgtt aatcccaacg 840 acggtagcct ttcctgacag ctgactgtaa attttgtcta acaaagaaaa aaaaagatta 900 gacatgtttt tccttgtcat tgattaggct ggatttcttt cagagtggaa cataggggat 960 atattggacc aaaagtagaa tgggtatata tttaaagtat ttctgataga acaggagtat 1020 attgtgcgaa aatatcctct attttctgtt gtctcctaat gagtttgaat gtaataatat 1080 tctcatgtgg acattgcttg caccaggttc tgtattaacc gaaccgcagg tttatggaag 1140 agacaaagag aaagatgaga tagtgaaaat cctaataaac aatgttagtg atgcccaaca 1200 cctttcagtc ctcccaatac ttggtatggg gggattagga aaaacgactc ttgcccaaat 1260 ggtcttcaat gaccagagag ttactgagca tttccattcc aaaatatgga tttgtgtctc 1320 ggaagatttt gatgagaaga ggttaataaa ggcaattgta gaatctattg aaggaaggcc 1380 actacttggt gagatggact tggctccact tcaaaagaag cttcaggagt tgctgaatgg 1440 aaaaagatac ttgcttgtct tagatgatgt ttggaatgaa gatcaacaga agtgggctaa 1500 tttaagagca gtcttgaagg ttggagcaag tggtgcttct gttctaacca ctactcgtct 1560 tgaaaaggtt ggatcaatta tgggaacatt gcaaccatat gaactgtcaa atctgtctca 1620 agaagattgt tggttgttgt tcatgcaacg tgcatttgga caccaagaag aaataaatcc 1680 aaaccttgtg gcaatcggaa aggagattgt gaaaaaaagt ggtggtgtgc ctctagcagc 1740 caaaactctt ggaggtattt tgtgcttcaa gagagaagaa agagcatggg aacatgtgag 1800 agacagtccg atttggaatt tgcctcaaga tgaaagttct attctgcctg ccctgaggct 1860 tagttaccat caacttccac ttgatttgaa acaatgcttt gcgtattgtg cggtgttccc 1920 aaaggatgcc aaaatggaaa aagaaaagct aatctctctc tggatggcgc atggttttct 1980 tttatcaaaa ggaaacatgg agctagagga tgtgggcgat gaagtatgga aagaattata 2040 cttgaggtct tttttccaag agattgaagt taaagatggt aaaacttatt tcaagatgca 2100 tgatctcatc catgatttgg caacatctct gttttcagca aacacatcaa gcagcaatat 2160 ccgtgaaata aataaacaca gttacacaca tatgatgtcc attggtttcg ccgaagtggt 2220 gtttttttac actcttcccc ccttggaaaa gtttatctcg ttaagagtgc ttaatctagg 2280 tgattcgaca tttaataagt taccatcttc cattggagat ctagtacatt taagatactt 2340 gaacctgtat ggcagtggca tgcgtagtct tccaaagcag ttatgcaagc ttcaaaatct 2400 gcaaactctt gatctacaat attgcaccaa gctttgttgt ttgccaaaag aaacaagtaa 2460 acttggtagt ctccgaaatc ttttacttga tggtagccag tcattgactt gtatgccacc 2520 aaggatagga tcattgacat gccttaagac tctaggtcaa tttgttgttg gaaggaagaa 2580 aggttatcaa cttggtgaac taggaaacct aaatctctat ggctcaatta aaatctcgca 2640 tcttgagaga gtgaagaatg ataaggacgc aaaagaagcc aatttatctg caaaagggaa 2700 tctgcattct ttaagcatga gttggaataa ctttggacca catatatatg aatcagaaga 2760 agttaaagtg cttgaagccc tcaaaccaca ctccaatctg acttctttaa aaatctatgg 2820 cttcagagga atccatctcc cagagtggat gaatcactca gtattgaaaa atattgtctc 2880 tattctaatt agcaacttca gaaactgctc atgcttacca ccctttggtg atctgccttg 2940 tctagaaagt ctagagttac actgggggtc tgcggatgtg gagtatgttg aagaagtgga 3000 tattgatgtt cattctggat tccccacaag aataaggttt ccatccttga ggaaacttga 3060 tatatgggac tttggtagtc tgaaaggatt gctgaaaaag gaaggagaag agcaattccc 3120 tgtgcttgaa gagatgataa ttcacgagtg cccttttctg accctttctt ctaatcttag 3180 ggctcttact tccctcagaa tttgctataa taaagtagct acttcattcc cagaagagat 3240 gttcaaaaac cttgcaaatc tcaaatactt gacaatctct cggtgcaata atctcaaaga 3300 gctgcctacc agcttggcta gtctgaatgc tttgaaaagt ctaaaaattc aattgtgttg 3360 cgcactagag agtctccctg aggaagggct ggaaggttta tcttcactca cagagttatt 3420 tgttgaacac tgtaacatgc taaaatgttt accagaggga ttgcagcacc taacaaccct 3480 cacaagttta aaaattcggg gatgtccaca actgatcaag cggtgtgaga agggaatagg 3540 agaagactgg cacaaaattt ctcacattcc taatgtgaat atatatattt aa 3592 50 5191 DNA Artificial Sequence Description of Artificial Sequence sequence of 5.2 kb Sca I genomic DNA fragment of S. bulbocastanum BAC SPB 4 present in pRGC2-blb 50 agtactccat ccgttcactt tgatttgtca tgttgcactt ttcgaaagtc aatttgacta 60 atttttaaag ctaaattaga ttacactaat tcaatatttt aaacagaaaa attagatatt 120 caaaaactat acaaaaaata ttatacattg caattttttg catatcaata tgataaaaaa 180 atatatcgta aaatattagt caaaattttt ataatttgac tcaaatcatg aaaagtataa 240 taattaatag tggacggagg aagtattgtc tttccagatt tgtggccatt tttggtccaa 300 gggccattag cagttctctt cattttctac ttctgtctca tattagatgg gcatcttact 360 aaaaatattt gtctcatatt acttgattat ttattaaatc aaaaagaatt aattaatttt 420 ttctcatttt acccctacaa ttaatatagt tttaaaagtt ttaaacaaat tttgaagaat 480 caaaatttct tttgcaagag acttattaat ataaacaaag gataaaataa taaaagctgt 540 caatttattg accatcactt aataatatat aaaatacaaa ctgctgatct aatatgagac 600 ggacaaaata tattctaaaa tattttcgga cagatatgtg atattctaac cattcactac 660 actatattat gcattttatc cgccaatgac ttatttcagc tttaattaat taggaaagag 720 gaaactgcca atgaggaaga gtaggggcgt agttgctgtc gacgaaaaaa agataatact 780 cactcttttc gatttttatt tttatttatc acttttaacc tatcatgtaa aaagataatt 840 atttttttca tgctttatcc ttagtattaa acaatttaat agggattatt ttgtaaaata 900 tttatatgaa taattgtttt cgtaatgaat ttgtccggtc aaacaatgat aaataaaaat 960 gaatgaagag agtagaaaac aaaacaaaag aacaagttga caacttgaga gattaaaagg 1020 gtccaaaacg ccttggattt tgagattcca tatgtgaaat ttccatgaaa taattgaatt 1080 tgtattatta caagtcaaac tttccatttc attccaacta gccatcttgg tttcaaaatt 1140 acacattcat tcattcacag atctaatatt cttaatagtg atttccacat atggctgaag 1200 ctttcattca agttctgcta gacaatctca cttctttcct caaaggggaa cttgtattgc 1260 ttttcggttt tcaagatgag ttccaaaggc tttcaagcat gttttctaca attcaagccg 1320 tccttgaaga tgctcaggag aagcaactca acaacaagcc tctagaaaat tggttgcaaa 1380 aactcaatgc tgctacatat gaagtcgatg acatcttgga tgaatataaa accaaggcca 1440 caagattctc ccagtctgaa tatggccgtt atcatccaaa ggttatccct ttccgtcaca 1500 aggtcgggaa aaggatggac caagtgatga aaaaactaaa ggcaattgct gaggaaagaa 1560 agaattttca tttgcacgaa aaaattgtag agagacaagc tgttagacgg gaaacaggta 1620 ctcatcttaa attagtatta caacaactaa gtttatattc atttttttgg caattatcaa 1680 attcagaaaa gggttaaata tactcatgtc ctatcgtaaa tagtgtatat atacctctcg 1740 ttgtactttc gatctgaata tacttgtcaa atctggcaag ctcagaatca aattatccac 1800 cccaactttt aaatactcga tatctttaga aatccacctg tctaactcat ccactaccca 1860 ttccctttgc tttgaattct tttctttacc tataaacttg gaacactcga tccgttttgc 1920 ttttcttaac aaagcagctc agagaaaaga ggttttcttc tattctgttt ctctgtgtgc 1980 tgcacttggg tccttaatcc cattaaaaac agggcatgtt aatcccaacg acggtagcct 2040 ttcctgacag ctgactgtaa attttgtcta acaaagaaaa aaaaagatta gacatgtttt 2100 tccttgtcat tgattaggct ggatttcttt cagagtggaa cataggggat atattggacc 2160 aaaagtagaa tgggtatata tttaaagtat ttctgataga acaggagtat attgtgcgaa 2220 aatatcctct attttctgtt gtctcctaat gagtttgaat gtaataatat tctcatgtgg 2280 acattgcttg caccaggttc tgtattaacc gaaccgcagg tttatggaag agacaaagag 2340 aaagatgaga tagtgaaaat cctaataaac aatgttagtg atgcccaaca cctttcagtc 2400 ctcccaatac ttggtatggg gggattagga aaaacgactc ttgcccaaat ggtcttcaat 2460 gaccagagag ttactgagca tttccattcc aaaatatgga tttgtgtctc ggaagatttt 2520 gatgagaaga ggttaataaa ggcaattgta gaatctattg aaggaaggcc actacttggt 2580 gagatggact tggctccact tcaaaagaag cttcaggagt tgctgaatgg aaaaagatac 2640 ttgcttgtct tagatgatgt ttggaatgaa gatcaacaga agtgggctaa tttaagagca 2700 gtcttgaagg ttggagcaag tggtgcttct gttctaacca ctactcgtct tgaaaaggtt 2760 ggatcaatta tgggaacatt gcaaccatat gaactgtcaa atctgtctca agaagattgt 2820 tggttgttgt tcatgcaacg tgcatttgga caccaagaag aaataaatcc aaaccttgtg 2880 gcaatcggaa aggagattgt gaaaaaaagt ggtggtgtgc ctctagcagc caaaactctt 2940 ggaggtattt tgtgcttcaa gagagaagaa agagcatggg aacatgtgag agacagtccg 3000 atttggaatt tgcctcaaga tgaaagttct attctgcctg ccctgaggct tagttaccat 3060 caacttccac ttgatttgaa acaatgcttt gcgtattgtg cggtgttccc aaaggatgcc 3120 aaaatggaaa aagaaaagct aatctctctc tggatggcgc atggttttct tttatcaaaa 3180 ggaaacatgg agctagagga tgtgggcgat gaagtatgga aagaattata cttgaggtct 3240 tttttccaag agattgaagt taaagatggt aaaacttatt tcaagatgca tgatctcatc 3300 catgatttgg caacatctct gttttcagca aacacatcaa gcagcaatat ccgtgaaata 3360 aataaacaca gttacacaca tatgatgtcc attggtttcg ccgaagtggt gtttttttac 3420 actcttcccc ccttggaaaa gtttatctcg ttaagagtgc ttaatctagg tgattcgaca 3480 tttaataagt taccatcttc cattggagat ctagtacatt taagatactt gaacctgtat 3540 ggcagtggca tgcgtagtct tccaaagcag ttatgcaagc ttcaaaatct gcaaactctt 3600 gatctacaat attgcaccaa gctttgttgt ttgccaaaag aaacaagtaa acttggtagt 3660 ctccgaaatc ttttacttga tggtagccag tcattgactt gtatgccacc aaggatagga 3720 tcattgacat gccttaagac tctaggtcaa tttgttgttg gaaggaagaa aggttatcaa 3780 cttggtgaac taggaaacct aaatctctat ggctcaatta aaatctcgca tcttgagaga 3840 gtgaagaatg ataaggacgc aaaagaagcc aatttatctg caaaagggaa tctgcattct 3900 ttaagcatga gttggaataa ctttggacca catatatatg aatcagaaga agttaaagtg 3960 cttgaagccc tcaaaccaca ctccaatctg acttctttaa aaatctatgg cttcagagga 4020 atccatctcc cagagtggat gaatcactca gtattgaaaa atattgtctc tattctaatt 4080 agcaacttca gaaactgctc atgcttacca ccctttggtg atctgccttg tctagaaagt 4140 ctagagttac actgggggtc tgcggatgtg gagtatgttg aagaagtgga tattgatgtt 4200 cattctggat tccccacaag aataaggttt ccatccttga ggaaacttga tatatgggac 4260 tttggtagtc tgaaaggatt gctgaaaaag gaaggagaag agcaattccc tgtgcttgaa 4320 gagatgataa ttcacgagtg cccttttctg accctttctt ctaatcttag ggctcttact 4380 tccctcagaa tttgctataa taaagtagct acttcattcc cagaagagat gttcaaaaac 4440 cttgcaaatc tcaaatactt gacaatctct cggtgcaata atctcaaaga gctgcctacc 4500 agcttggcta gtctgaatgc tttgaaaagt ctaaaaattc aattgtgttg cgcactagag 4560 agtctccctg aggaagggct ggaaggttta tcttcactca cagagttatt tgttgaacac 4620 tgtaacatgc taaaatgttt accagaggga ttgcagcacc taacaaccct cacaagttta 4680 aaaattcggg gatgtccaca actgatcaag cggtgtgaga agggaatagg agaagactgg 4740 cacaaaattt ctcacattcc taatgtgaat atatatattt aagttatttg ctattgtttc 4800 tttgtttgtg agtctttttg gttcctgcca ttgtgattgc atgtaatttt tttctagggt 4860 tgtttcttta tgagtctctc tctcattgga tgtaattttc ttttggaaac aaatctgtca 4920 attgatttgt attatacgct ttcagaatct attacttatt tgtaattgtt tctttgtttg 4980 taaattgtga gtatcttatt ttatggaatt ttctgatttt attttgaaaa caaatcaatg 5040 atttgtaaga tccatctgta ttatactccc ttcgtctcat tttatgtgtc acctgtcgga 5100 tttcgagatt caaacaaatc tatctttgat cgtaaatttt taatagatct tttaaacatt 5160 ttgaattatc aattattgtg actttagtac t 5191 51 3260 DNA Solanum bulbocastanum misc_feature (1)..(3260) /note=“RGC1-blb” 51 atggctgaag ctttccttca agttctgcta gataatctca cttttttcat ccaaggggaa 60 cttggattgg tttttggttt cgagaaggag tttaaaaaac tttcaagtat gttttcaatg 120 atccaagctg tgctagaaga tgctcaagag aagcaactga agtacaaggc aataaagaac 180 tggttacaga aactcaatgt tgctgcatat gaagttgatg acatcttgga tgactgtaaa 240 actgaggcag caagattcaa gcaggctgta ttggggcgtt atcatccacg gaccatcact 300 ttctgttaca aggtgggaaa aagaatgaaa gaaatgatgg aaaaactaga tgcaattgca 360 gaggaacgga ggaattttca tttagatgaa aggattatag agagacaagc tgctagacgg 420 caaacaggtg ctcatcttaa ttttatttta aaacaaataa gtattacaaa ttgcagagaa 480 acgaaggaat ttatattcat ttttattttt ggcaattatc aaagtcattt gtgtttttaa 540 gctgggggga agtttcaaat attttctcta gtcttaatgt ttgtctcact cactcagcat 600 gattttctca atccttcact tcaactcccc cctactgtgc aaatatcttc tctattttct 660 gttgactcct aatgagcttg aatgtaacaa cattcttgtt tggagcaggt tttgttttaa 720 ctgagccaaa agtttatgga agggaaaaag aggaggatga gatagtgaaa atcttgataa 780 acaatgttag ttattccgaa gaagttccag tactcccaat acttggtatg gggggactag 840 gaaagacgac tctagcccaa atggtcttca atgatcaaag aattactgag catttcaatc 900 taaagatatg ggtttgtgtc tcagatgatt ttgatgagaa gaggttgatt aaggcaattg 960 tagaatctat tgaaggaaag tcactgggtg acatggactt ggctcccctc cagaaaaagc 1020 ttcaggagtt gttgaatgga aaaagatact ttcttgtttt ggatgatgtt tggaatgaag 1080 atcaagaaaa gtgggataat cttagagcag tattgaagat tggagctagt ggtgcttcaa 1140 ttctaattac tactcgtctt gaaaaaattg gatcaattat gggaactttg caactatatc 1200 agttatcaaa tttgtctcaa gaagattgtt ggttgttgtt caagcaacgt gcattttgcc 1260 accaaaccga aacaagtcct aaacttatgg aaatcggaaa ggagattgtg aagaaatgtg 1320 ggggtgtgcc tctagcagcc aaaactcttg gaggcctttt acgcttcaag agggaagaaa 1380 gtgaatggga acatgtgaga gatagtgaga tttggaattt acctcaagat gaaaattctg 1440 ttttgcctgc cctgaggctg agttatcatc atcttccact tgatttgaga caatgttttg 1500 catattgcgc agtattccca aaggacacca aaatagaaaa ggaatatctc atcgctctct 1560 ggatggcaca cagttttctt ttatcaaaag gaaacatgga gctagaggat gtgggcaatg 1620 aagtatggaa tgaattatac ttgaggtctt ttttccaaga gattgaagtt aaatctggta 1680 aaacttattt caagatgcat gatctcatcc atgatttggc tacatctatg ttttcagcaa 1740 gcgcatcaag cagaagtata cgccaaataa atgtaaaaga tgatgaagat atgatgttca 1800 ttgtaacaaa ttataaagat atgatgtcca ttggtttctc cgaagtggtg tcttcttact 1860 ctccttcgct ctttaaaagg tttgtctcgt taagggtgct taatctaagt aactcagaat 1920 ttgaacagtt accgtcttcc gttggagatc tagtacattt aagatacctt gacctgtctg 1980 gtaataaaat ttgtagtctt ccaaagaggt tgtgcaagct tcaaaatctg cagactcttg 2040 atctatataa ttgccagtca ctttcttgtt tgccgaaaca aacaagtaag ctttgtagtc 2100 tccggaatct tgtacttgat cactgtccat tgacttctat gccaccaaga ataggattgt 2160 tgacatgcct taagacacta ggttactttg ttgtaggcga gaggaaaggt tatcaacttg 2220 gtgaactacg aaatttaaac ctccgtggtg caatttcaat cacacatctt gagagagtga 2280 aaaatgatat ggaggcaaaa gaagccaatt tatctgcaaa agcaaatcta cactctttaa 2340 gcatgagttg ggatagacca aacagatatg aatccgaaga agttaaagtg cttgaagccc 2400 tcaaaccaca tcccaatctg aaatatttag aaatcattga cttctgtgga ttctgtctcc 2460 ctgactggat gaatcactca gttttgaaaa atgttgtctc tattctaatt agcggttgtg 2520 aaaactgctc gtgcttacca ccctttggtg agctgccttg tctagaaagt ctggagttac 2580 aagacgggtc tgtggaggtg gagtatgttg aagattctgg attcctgaca agaagaagat 2640 ttccatccct gagaaaactt catataggtg gcttttgtaa tctgaaagga ttgcagagaa 2700 tgaaaggagc agagcaattc cccgtgcttg aagagatgaa gatttcggat tgccctatgt 2760 ttgtttttcc gaccctttct tctgtcaaga aattagaaat ttggggggag gcagatgcag 2820 gaggtttgag ctccatatct aatctcagca ctcttacatc cctcaagatt ttcagtaacc 2880 acacagtgac ttcactactg gaagagatgt tcaaaaacct tgaaaatctc atatacttga 2940 gtgtctcttt cttggagaat ctcaaagagc tgcctaccag cctggctagt ctcaacaatt 3000 tgaagtgtct ggatattcgt tattgttacg cactagagag tctccccgag gaagggctgg 3060 aaggtttatc ttcactcaca gagttatttg ttgaacactg taacatgcta aaatgtttac 3120 cagagggatt gcagcaccta acaaccctca caagtttaaa aattcgggga tgtccacaac 3180 tgatcaagcg gtgtgagaag ggaataggag aagactggca caaaatttct cacattccta 3240 atgtgaatat atatatttaa 3260 52 3971 DNA Solanum bulbocastanum misc_feature (1)..(3971) /note=“RGC3-blb” 52 atggctgaag ctttcattca agttgtgcta gacaatctca cttctttcct caaaggggaa 60 cttgtattgc ttttcggttt tcaagatgag ttccaaaggc tttcaagcat gttttctaca 120 atccaagccg tccttgaaga tgctcaagag aagcaactca acgacaagcc tctagaaaat 180 tggttgcaaa aactcaatgc tgctacatat gaagtcgatg acatcttgga tgaatataaa 240 actaaggcca caagattctt gcagtctgaa tatggccgtt atcatccaaa ggttatccct 300 ttccgtcaca aggttgggaa aaggatggac caagtgatga aaaaactgaa tgcaattgct 360 gaggaacgaa agaattttca tttgcaagaa aagattatag agagacaagc tgctacacgg 420 gaaacaggta ctcatcttaa attagtatta caacttagtt tatattcatt tgttttgggc 480 aatgatcaaa ttatgtaaag gtcaaatata ctcatgtact actgaaaata gtttaaatat 540 acctctagtt atactattag tacgaacata ctcctcccat atactttgga acaaatattc 600 ccttaacgaa ataagacacg tgaaaagttc agattcaaat tatccaccct caattttaag 660 atctgatttc tttaggaaac cactcatctc ctccgttttg agttcttaac gaagcagctc 720 agagaaaaga ggttttcttc tgttctgttt ctgctgcatt tgtgtcttaa tccaataaca 780 aacaatacaa attaatatta tgttcacgat gagggtagtc tttctagcta gacatgaact 840 gagtgtaaat tttgttttaa ggaagaaaaa gaaatgatta ggctggattt ctttcagagt 900 ggaatatagg gggataaagt tggagcatag agttccatcg tttatttctt tccttaaagt 960 aacaagttca acaaaatgat atcaaggtac ggtaatggaa aattattaga cacgtctaaa 1020 ctacaaaaat ggaatagaaa cttaaattat cagtgacaat atcatccttt aataaagcta 1080 ccaaatttaa atcatgatac agagaagaaa ccaaaaaaat taggggtgaa ttatttgatt 1140 ctatgcttat cacatgtctt cccatcaaca tcaaaggaaa aattgtgcca aagtataaac 1200 ggtgcggtat atttggattg aaagtaaaac aggaggatac atttggacta aaagtataac 1260 aataagtata tttgatcatt ttatgtatca aattcatgtg gtttttgggg agaagggaag 1320 tttcaatgtt ttcaatctgc tcctcatctc atccatatct ctttattgtg caaaaccctt 1380 ctctatttaa ctattttctg ccgactccta atgagcttga atgtaacaat attctcatct 1440 ggacattgct tgcaccaggt tctgtgttaa ctgaaccaca agtttatgga agggacaaag 1500 aaaaagatga gatagtgaaa atcctaataa acaatgttag tgatgcccaa aaactctcag 1560 tcctcccaat acttggtatg gggggactag gaaagacaac tctttcccaa atggtcttca 1620 atgatcagag agtaactgag cgtttctatc ccaaaatatg gatttgcgtc tcggatgatt 1680 ttgatgagaa gaggttgata aaggcaatag tagaatctat tgaagggaag tccctcagtg 1740 acatggactt ggctccactt caaaagaagc ttcaagagtt gctgaatgga aaaagatact 1800 tccttgtctt agatgatgtt tggaatgaag atcaacataa gtgggctaat ttaagagcag 1860 tcttgaaggt tggagcaagt ggtgcatttg ttctaactac tactcgtctt gaaaaggttg 1920 gatcaattat gggaacattg caaccatatg aattgtcaaa tctgtctcca gaggattgtt 1980 ggtttttgtt catgcagcgt gcatttggac accaagaaga aataaatcca aaccttgtgg 2040 caatcggaaa ggagattgtg aaaaaatgtg gtggtgtgcc tctagcagcc aagactcttg 2100 gaggtatttt gcgcttcaag agagaagaaa gagaatggga acatgtgaga gacagtccga 2160 tttggaattt gcctcaagat gaaagttcta ttctgcctgc cctgaggctt agttaccatc 2220 atcttccact tgatttgaga caatgctttg tgtattgtgc ggtattccca aaggacacca 2280 aaatggcaaa ggaaaatctt atcgcttttt ggatggcaca tggttttctt ttatcgaaag 2340 gaaatttgga gctagaggat gtaggtaatg aagtatggaa tgaattatac ttgaggtctt 2400 tcttccaaga gattgaagtt gaatctggta aaacttattt caagatgcat gacctcatcc 2460 atgatttggc tacatctctg ttttcagcaa acacatcaag cagcaatatt cgtgaaataa 2520 atgctaatta tgatggatat atgatgtcga ttggttttgc tgaagtggta tcttcttact 2580 ctccttcact cttgcaaaag tttgtctcat taagggtgct taatctaaga aactcgaacc 2640 taaatcaatt accatcttcc attggagatc tagtacattt aagatacctg gacttgtctg 2700 gcaattttag aattcgtaat cttccaaaga gattatgcag gcttcaaaat ctgcagactc 2760 ttgatctaca ttattgcgac tctctttctt gtttgccaaa acaaacaagt aaacttggta 2820 gtctccgaaa tcttttactt gatggctgtt cattgacgtc aacgccacca aggataggat 2880 tgttgacatg ccttaagtct ctaagttgct ttgttattgg caagagaaaa ggttatcaac 2940 ttggtgaact aaaaaaccta aatctctatg gctcaatttc aatcacaaaa cttgacagag 3000 tgaagaaaga tagcgatgca aaagaagcta atttatctgc taaagcaaat ctgcactctt 3060 tatgcctgag ttgggacctt gatggaaaac atagatatga ttcagaagtt cttgaagccc 3120 tcaaaccaca ctccaatctg aaatatttag aaatcaatgg cttcggagga atccgtctcc 3180 cagattggat gaatcaatca gttttgaaaa atgttgtctc tattagaatt agaggttgtg 3240 aaaactgctc atgcttacca ccctttggtg agctgccttg tctagaaagt ctagagttac 3300 acaccgggtc agcagatgtg gagtatgttg aagataatgt tcatcctgga aggtttccat 3360 ccttgaggaa acttgttata tgggacttta gtaatctaaa aggattgctg aaaaaggaag 3420 gagaaaagca attccctgtg cttgaagaga tgacatttta ctggtgccct atgtttgtta 3480 ttccgaccct ttcttctgtc aagacattga aagttattgc gacagatgca acagttttga 3540 ggtccatatc taatcttagg gctcttactt cccttgacat tagcaataac gtagaagcta 3600 cttcactccc agaagagatg ttcaaaagcc ttgcaaatct caaatacttg aatatctctt 3660 tctttaggaa tctcaaagag ttgcctacca gcctggctag tctcaatgct ttgaagagtc 3720 tcaaatttga attttgtaac gcactagaga gtctcccaga ggaaggggtg aaaggtttaa 3780 cttcactcac cgagttgtct gtcagtaact gtatgatgct aaaatgttta ccggagggat 3840 tgcagcacct aacagccctc acaactttaa caattactca atgtccaata gtattcaagc 3900 ggtgtgagag aggaatagga gaagactggc acaaaattgc tcacattcca tatttgactc 3960 tatatgagtg a 3971 53 3899 DNA Solanum bulbocastanum misc_feature (1)..(3899) /note=“RGC4-blb” 53 atggcggaag cttttcttca agttctgcta gaaaatctca cttctttcat cggagataaa 60 cttgtattga ttttcggttt cgaaaaggaa tgtgaaaagc tgtcgagtgt gttttccaca 120 attcaagctg tgcttcaaga tgctcaggag aagcaattga aggacaaggc aattgagaat 180 tggttgcaga aactcaattc tgctgcctat gaagttgatg atatattggg cgaatgtaaa 240 aatgaggcaa taagatttga gcagtctcga ttagggtttt atcacccagg gattatcaat 300 ttccgtcaca aaattgggag aaggatgaaa gagataatgg agaaactaga tgcaatatct 360 gaggaaagaa ggaagtttca tttccttgaa aaaattacag agagacaagc tgccgctgct 420 acgcgtgaaa caggtgtgag tactgagtaa ttgtagctta gttaatattc aatttgttac 480 cacatcatgt gttcaccgtg atctctacag taggatggca atggggctgg gcgaggttgg 540 aggtgtgcag gtgtgtggcg caaccccaac tttgagtcta cataagtagg tacttaaatt 600 tgtatagagt tgaacaagta caaacgcctc ctacttggtg tccttatgcg tattatgtca 660 cttaggatgc atgtgtctac ttgttcaact ttatatgagt ttaagttcta cttgtgcaca 720 cccaaagttg gagcgcgtag atgtcagttg ataccaagtt aaaaaggcat atttatgaat 780 tatgccttta aattatgatt caattttgta tcagtctgtc caaaatatgt tctagtgaaa 840 gtgttaaact tagtctggat ctgctattga aagtgaattt ttgtggcact aaacaatgca 900 atgggtctgg attcattttt gcattaactt ttgtttagac gattttcttt atcgaatttt 960 actgtctaaa atggaaaaag caaagaaata agaagtatac agaggctgac ttcttcatag 1020 tatctatcat ataaaaaaaa gcattgatta ctaggatatg ggttctttta aattacaaat 1080 ttgtgagtta aaacagttct gttgggaagg atttagatac acgtggatag tatctagaag 1140 ttttttaaat aaaaaattag caaattatgc gggctggggc gggttgaaaa cagcaaactt 1200 tgcaaggctt ggcgggtcga aatctttgca agtttgtgtg ggtttgccct gcaccaccca 1260 atctgccatt cctgtctaaa tgtttgtttt gtctataatt cttgctgact cattctaatg 1320 agctcaattg taacaaattc tttgtgtcca cattacttgg aacaggtttt gtgttaactg 1380 aaccaaaagt ctacggaagg gacaaagagg aggatgagat agtgaaaatt ctgataaaca 1440 atgttaatgt tgccgaagaa cttccagtct tccctataat tggtatgggg ggactaggaa 1500 agacgacact tgcccaaatg atcttcaacg atgagagagt aactaagcat ttcaatccca 1560 aaatatgggt ttgtgtctca gatgattttg atgagaagag gttaattaag acaattatag 1620 gaaatattga aagaagttct cctcatgttg aggacttggc ttcatttcag aagaagctcc 1680 aggagttatt gaatggaaaa cgatacttgc ttgtcttaga tgatgtttgg aatgatgatc 1740 tagaaaagtg ggctaagtta agagcagtct taactgttgg agcaagaggt gcttctattc 1800 tagctactac tcgtcttgaa aaggttggat caattatggg aacgttgcaa ccatatcatt 1860 tgtcaaattt gtctccacat gatagtttac ttttgtttat gcaacgcgca tttgggcaac 1920 aaaaagaagc aaatcctaat ctagtggcca ttggaaagga gattgtgaag aaatgtggtg 1980 gtgtgccttt agcagccaag actcttggtg gtcttttacg cttcaagaga gaagagagtg 2040 aatgggaaca tgtgagagat aatgagattt ggagtctgcc tcaagatgaa agttctattt 2100 tgcctgctct aagactgagt tatcatcacc ttccacttga tttgagacaa tgctttgcgt 2160 attgtgcagt attcccaaag gacaccaaaa tgataaagga aaatctcatt actctctgga 2220 tggcgcatgg ttttctttta tcaaagggaa acttggagct agaggatgtg ggtaatgaag 2280 tatggaatga attatacttg aggtctttct tccaagaaat tgaagctaaa tcgggtaata 2340 cttatttcaa gatacatgat ctaatccatg atttggctac atctctgttt tcggcaagcg 2400 catcatgcgg caatatccgc gaaataaatg tcaaagatta taagcataca gtgtccattg 2460 gtttcgctgc agtggtgtct tcttactctc cttcgctctt gaaaaagttt gtctcgttaa 2520 gggtgcttaa tctaagttac tcaaaacttg agcaattacc gtcttccatt ggagatctat 2580 tacatttaag atacctggac ctgtcttgca ataacttccg tagtcttcca gagaggttgt 2640 gcaagcttca aaatcttcag actcttgatg tacataattg ctactcactt aattgtttgc 2700 caaaacaaac aagtaaactt agtagtctcc gacatcttgt tgttgatggc tgtccattga 2760 cttctactcc accaaggata ggattgttga catgccttaa gactctaggt ttctttattg 2820 tgggaagcaa gaaaggttat caacttggtg aactgaaaaa cctaaatctc tgcggctcaa 2880 tttcaatcac acaccttgag agagtgaaga acgatacgga tgcagaagcc aatttatctg 2940 caaaagcaaa tctgcaatct ttaagcatga gttgggataa cgatggacca aacagatatg 3000 aatccaaaga agttaaagtg cttgaagcac tcaaaccaca ccccaatctg aaatatttag 3060 agatcattgc cttcggagga ttccgttttc caagctggat aaatcactca gttttggaga 3120 aggtcatctc tgttagaatt aaaagctgca aaaactgctt gtgcttacca ccctttgggg 3180 agcttccttg tctagaaaat ctagagttac aaaacggatc tgcggaggtg gagtatgttg 3240 aagaggatga tgtccattct agattctcca caagaagaag ctttccatcc ctgaaaaaac 3300 ttcgtatatg gttctttcgc agtttgaaag ggctgatgaa agaggaagga gaagagaaat 3360 tccccatgct tgaagagatg gcgattttat attgccctct gtttgttttt ccaacccttt 3420 cttctgtcaa gaaattagaa gttcacggca acacaaacac tagaggtttg agctccatat 3480 ctaatcttag cactcttact tccctccgca ttggtgctaa ctacagagcg acttcactcc 3540 cagaagagat gttcacaagt cttacaaatc tcgaattctt gagtttcttt gacttcaaga 3600 atctcaaaga tctgcctacc agcctgacta gtctcaatgc tttgaagcgt ctccaaattg 3660 aaagttgtga ctcactagag agtttccctg aacaagggct agaaggttta acttcactca 3720 cacagttgtt tgttaaatac tgtaagatgc taaaatgttt acccgaggga ttgcagcacc 3780 taacagccct cacaaattta ggagtttctg gttgtccaga agtggaaaag cgctgtgata 3840 aggaaatagg agaagactgg cacaaaattg ctcacattcc aaatctggat attcattag 3899 54 970 PRT Artificial Sequence Description of Artificial Sequence deduced Rpi-blb protein sequence domain A, B and C 54 Met Ala Glu Ala Phe Ile Gln Val Leu Leu Asp Asn Leu Thr Ser Phe 1 5 10 15 Leu Lys Gly Glu Leu Val Leu Leu Phe Gly Phe Gln Asp Glu Phe Gln 20 25 30 Arg Leu Ser Ser Met Phe Ser Thr Ile Gln Ala Val Leu Glu Asp Ala 35 40 45 Gln Glu Lys Gln Leu Asn Asn Lys Pro Leu Glu Asn Trp Leu Gln Lys 50 55 60 Leu Asn Ala Ala Thr Tyr Glu Val Asp Asp Ile Leu Asp Glu Tyr Lys 65 70 75 80 Thr Lys Ala Thr Arg Phe Ser Gln Ser Glu Tyr Gly Arg Tyr His Pro 85 90 95 Lys Val Ile Pro Phe Arg His Lys Val Gly Lys Arg Met Asp Gln Val 100 105 110 Met Lys Lys Leu Lys Ala Ile Ala Glu Glu Arg Lys Asn Phe His Leu 115 120 125 His Glu Lys Ile Val Glu Arg Gln Ala Val Arg Arg Glu Thr Gly Ser 130 135 140 Val Leu Thr Glu Pro Gln Val Tyr Gly Arg Asp Lys Glu Lys Asp Glu 145 150 155 160 Ile Val Lys Ile Leu Ile Asn Asn Val Ser Asp Ala Gln His Leu Ser 165 170 175 Val Leu Pro Ile Leu Gly Met Gly Gly Leu Gly Lys Thr Thr Leu Ala 180 185 190 Gln Met Val Phe Asn Asp Gln Arg Val Thr Glu His Phe His Ser Lys 195 200 205 Ile Trp Ile Cys Val Ser Glu Asp Phe Asp Glu Lys Arg Leu Ile Lys 210 215 220 Ala Ile Val Glu Ser Ile Glu Gly Arg Pro Leu Leu Gly Glu Met Asp 225 230 235 240 Leu Ala Pro Leu Gln Lys Lys Leu Gln Glu Leu Leu Asn Gly Lys Arg 245 250 255 Tyr Leu Leu Val Leu Asp Asp Val Trp Asn Glu Asp Gln Gln Lys Trp 260 265 270 Ala Asn Leu Arg Ala Val Leu Lys Val Gly Ala Ser Gly Ala Ser Val 275 280 285 Leu Thr Thr Thr Arg Leu Glu Lys Val Gly Ser Ile Met Gly Thr Leu 290 295 300 Gln Pro Tyr Glu Leu Ser Asn Leu Ser Gln Glu Asp Cys Trp Leu Leu 305 310 315 320 Phe Met Gln Arg Ala Phe Gly His Gln Glu Glu Ile Asn Pro Asn Leu 325 330 335 Val Ala Ile Gly Lys Glu Ile Val Lys Lys Ser Gly Gly Val Pro Leu 340 345 350 Ala Ala Lys Thr Leu Gly Gly Ile Leu Cys Phe Lys Arg Glu Glu Arg 355 360 365 Ala Trp Glu His Val Arg Asp Ser Pro Ile Trp Asn Leu Pro Gln Asp 370 375 380 Glu Ser Ser Ile Leu Pro Ala Leu Arg Leu Ser Tyr His Gln Leu Pro 385 390 395 400 Leu Asp Leu Lys Gln Cys Phe Ala Tyr Cys Ala Val Phe Pro Lys Asp 405 410 415 Ala Lys Met Glu Lys Glu Lys Leu Ile Ser Leu Trp Met Ala His Gly 420 425 430 Phe Leu Leu Ser Lys Gly Asn Met Glu Leu Glu Asp Val Gly Asp Glu 435 440 445 Val Trp Lys Glu Leu Tyr Leu Arg Ser Phe Phe Gln Glu Ile Glu Val 450 455 460 Lys Asp Gly Lys Thr Tyr Phe Lys Met His Asp Leu Ile His Asp Leu 465 470 475 480 Ala Thr Ser Leu Phe Ser Ala Asn Thr Ser Ser Ser Asn Ile Arg Glu 485 490 495 Ile Asn Lys His Ser Tyr Thr His Met Met Ser Ile Gly Phe Ala Glu 500 505 510 Val Val Phe Phe Tyr Thr Leu Pro Pro Leu Glu Lys Phe Ile Ser Leu 515 520 525 Arg Val Leu Asn Leu Gly Asp Ser Thr Phe Asn Lys Leu Pro Ser Ser 530 535 540 Ile Gly Asp Leu Val His Leu Arg Tyr Leu Asn Leu Tyr Gly Ser Gly 545 550 555 560 Met Arg Ser Leu Pro Lys Gln Leu Cys Lys Leu Gln Asn Leu Gln Thr 565 570 575 Leu Asp Leu Gln Tyr Cys Thr Lys Leu Cys Cys Leu Pro Lys Glu Thr 580 585 590 Ser Lys Leu Gly Ser Leu Arg Asn Leu Leu Leu Asp Gly Ser Gln Ser 595 600 605 Leu Thr Cys Met Pro Pro Arg Ile Gly Ser Leu Thr Cys Leu Lys Thr 610 615 620 Leu Gly Gln Phe Val Val Gly Arg Lys Lys Gly Tyr Gln Leu Gly Glu 625 630 635 640 Leu Gly Asn Leu Asn Leu Tyr Gly Ser Ile Lys Ile Ser His Leu Glu 645 650 655 Arg Val Lys Asn Asp Lys Asp Ala Lys Glu Ala Asn Leu Ser Ala Lys 660 665 670 Gly Asn Leu His Ser Leu Ser Met Ser Trp Asn Asn Phe Gly Pro His 675 680 685 Ile Tyr Glu Ser Glu Glu Val Lys Val Leu Glu Ala Leu Lys Pro His 690 695 700 Ser Asn Leu Thr Ser Leu Lys Ile Tyr Gly Phe Arg Gly Ile His Leu 705 710 715 720 Pro Glu Trp Met Asn His Ser Val Leu Lys Asn Ile Val Ser Ile Leu 725 730 735 Ile Ser Asn Phe Arg Asn Cys Ser Cys Leu Pro Pro Phe Gly Asp Leu 740 745 750 Pro Cys Leu Glu Ser Leu Glu Leu His Trp Gly Ser Ala Asp Val Glu 755 760 765 Tyr Val Glu Glu Val Asp Ile Asp Val His Ser Gly Phe Pro Thr Arg 770 775 780 Ile Arg Phe Pro Ser Leu Arg Lys Leu Asp Ile Trp Asp Phe Gly Ser 785 790 795 800 Leu Lys Gly Leu Leu Lys Lys Glu Gly Glu Glu Gln Phe Pro Val Leu 805 810 815 Glu Glu Met Ile Ile His Glu Cys Pro Phe Leu Thr Leu Ser Ser Asn 820 825 830 Leu Arg Ala Leu Thr Ser Leu Arg Ile Cys Tyr Asn Lys Val Ala Thr 835 840 845 Ser Phe Pro Glu Glu Met Phe Lys Asn Leu Ala Asn Leu Lys Tyr Leu 850 855 860 Thr Ile Ser Arg Cys Asn Asn Leu Lys Glu Leu Pro Thr Ser Leu Ala 865 870 875 880 Ser Leu Asn Ala Leu Lys Ser Leu Lys Ile Gln Leu Cys Cys Ala Leu 885 890 895 Glu Ser Leu Pro Glu Glu Gly Leu Glu Gly Leu Ser Ser Leu Thr Glu 900 905 910 Leu Phe Val Glu His Cys Asn Met Leu Lys Cys Leu Pro Glu Gly Leu 915 920 925 Gln His Leu Thr Thr Leu Thr Ser Leu Lys Ile Arg Gly Cys Pro Gln 930 935 940 Leu Ile Lys Arg Cys Glu Lys Gly Ile Gly Glu Asp Trp His Lys Ile 945 950 955 960 Ser His Ile Pro Asn Val Asn Ile Tyr Ile 965 970 55 979 PRT Artificial Sequence Description of Artificial Sequence alignment RGC3-blb 55 Met Ala Glu Ala Phe Ile Gln Val Val Leu Asp Asn Leu Thr Ser Phe 1 5 10 15 Leu Lys Gly Glu Leu Val Leu Leu Phe Gly Phe Gln Asp Glu Phe Gln 20 25 30 Arg Leu Ser Ser Met Phe Ser Thr Ile Gln Ala Val Leu Glu Asp Ala 35 40 45 Gln Glu Lys Gln Leu Asn Asp Lys Pro Leu Glu Asn Trp Leu Gln Lys 50 55 60 Leu Asn Ala Ala Thr Tyr Glu Val Asp Asp Ile Leu Asp Glu Tyr Lys 65 70 75 80 Thr Lys Ala Thr Arg Phe Leu Gln Ser Glu Tyr Gly Arg Tyr His Pro 85 90 95 Lys Val Ile Pro Phe Arg His Lys Val Gly Lys Arg Met Asp Gln Val 100 105 110 Met Lys Lys Leu Asn Ala Ile Ala Glu Glu Arg Lys Asn Phe His Leu 115 120 125 Gln Glu Lys Ile Ile Glu Arg Gln Ala Ala Thr Arg Glu Thr Gly Ser 130 135 140 Val Leu Thr Glu Pro Gln Val Tyr Gly Arg Asp Lys Glu Lys Asp Glu 145 150 155 160 Ile Val Lys Ile Leu Ile Asn Asn Val Ser Asp Ala Gln Lys Leu Ser 165 170 175 Val Leu Pro Ile Leu Gly Met Gly Gly Leu Gly Lys Thr Thr Leu Ser 180 185 190 Gln Met Val Phe Asn Asp Gln Arg Val Thr Glu Arg Phe Tyr Pro Lys 195 200 205 Ile Trp Ile Cys Val Ser Asp Asp Phe Asp Glu Lys Arg Leu Ile Lys 210 215 220 Ala Ile Val Glu Ser Ile Glu Gly Lys Ser Leu Ser Asp Met Asp Leu 225 230 235 240 Ala Pro Leu Gln Lys Lys Leu Gln Glu Leu Leu Asn Gly Lys Arg Tyr 245 250 255 Phe Leu Val Leu Asp Asp Val Trp Asn Glu Asp Gln His Lys Trp Ala 260 265 270 Asn Leu Arg Ala Val Leu Lys Val Gly Ala Ser Gly Ala Phe Val Leu 275 280 285 Thr Thr Thr Arg Leu Glu Lys Val Gly Ser Ile Met Gly Thr Leu Gln 290 295 300 Pro Tyr Glu Leu Ser Asn Leu Ser Pro Glu Asp Cys Trp Phe Leu Phe 305 310 315 320 Met Gln Arg Ala Phe Gly His Gln Glu Glu Ile Asn Pro Asn Leu Val 325 330 335 Ala Ile Gly Lys Glu Ile Val Lys Lys Cys Gly Gly Val Pro Leu Ala 340 345 350 Ala Lys Thr Leu Gly Gly Ile Leu Arg Phe Lys Arg Glu Glu Arg Glu 355 360 365 Trp Glu His Val Arg Asp Ser Pro Ile Trp Asn Leu Pro Gln Asp Glu 370 375 380 Ser Ser Ile Leu Pro Ala Leu Arg Leu Ser Tyr His His Leu Pro Leu 385 390 395 400 Asp Leu Asp Gln Cys Phe Val Tyr Cys Ala Val Phe Pro Lys Asp Thr 405 410 415 Lys Met Ala Lys Glu Asn Leu Ile Ala Phe Trp Met Ala His Gly Phe 420 425 430 Leu Leu Ser Lys Gly Asn Leu Glu Leu Glu Asp Val Gly Asn Glu Val 435 440 445 Trp Asn Glu Leu Tyr Leu Arg Ser Phe Phe Gln Glu Ile Glu Val Glu 450 455 460 Ser Gly Lys Thr Tyr Phe Lys Met His Asp Leu Ile His Asp Leu Ala 465 470 475 480 Thr Ser Leu Phe Ser Ala Asn Thr Ser Ser Ser Asn Ile Arg Glu Ile 485 490 495 Asn Ala Asn Tyr Asp Gly Tyr Met Met Ser Ile Gly Phe Ala Glu Val 500 505 510 Val Ser Ser Tyr Ser Pro Ser Leu Leu Gln Lys Phe Val Ser Leu Arg 515 520 525 Val Leu Asn Leu Arg Asn Ser Asn Leu Asn Gln Leu Pro Ser Ser Ile 530 535 540 Gly Asp Leu Val His Leu Arg Tyr Leu Asp Leu Ser Gly Asn Phe Arg 545 550 555 560 Ile Arg Asn Leu Pro Lys Arg Leu Cys Lys Leu Gln Asn Leu Gln Thr 565 570 575 Leu Asp Leu His Tyr Cys Asp Ser Leu Ser Cys Leu Pro Lys Gln Thr 580 585 590 Ser Lys Leu Gly Ser Leu Arg Asn Leu Leu Leu Asp Gly Cys Ser Leu 595 600 605 Thr Ser Thr Pro Pro Arg Ile Gly Leu Leu Thr Cys Leu Lys Ser Leu 610 615 620 Ser Cys Phe Val Ile Gly Lys Arg Lys Gly Tyr Gln Leu Gly Glu Leu 625 630 635 640 Lys Asn Leu Asn Leu Tyr Gly Ser Ile Ser Ile Thr Lys Leu Asp Arg 645 650 655 Val Lys Lys Asp Ser Asp Ala Lys Glu Ala Asn Leu Ser Ala Lys Ala 660 665 670 Asn Leu His Ser Leu Cys Leu Ser Trp Asp Leu Asp Gly Lys His Arg 675 680 685 Tyr Asp Ser Glu Val Leu Glu Ala Leu Lys Pro His Ser Asn Leu Lys 690 695 700 Tyr Leu Glu Ile Asn Gly Phe Gly Gly Ile Arg Leu Pro Asp Trp Met 705 710 715 720 Asn Gln Ser Val Leu Lys Asn Val Val Ser Ile Arg Ile Arg Gly Cys 725 730 735 Glu Asn Cys Ser Cys Leu Pro Pro Phe Gly Glu Leu Pro Cys Leu Glu 740 745 750 Ser Leu Glu Leu His Thr Gly Ser Ala Asp Val Glu Tyr Val Glu Asp 755 760 765 Asn Val His Pro Gly Arg Phe Pro Ser Leu Arg Lys Leu Val Ile Trp 770 775 780 Asp Phe Ser Asn Leu Lys Gly Leu Leu Lys Lys Glu Gly Glu Glu Gln 785 790 795 800 Phe Pro Val Leu Glu Glu Met Thr Phe Tyr Trp Cys Pro Met Phe Val 805 810 815 Ile Pro Thr Leu Ser Ser Val Lys Thr Leu Lys Val Ile Ala Thr Asp 820 825 830 Ala Thr Val Leu Arg Ser Ile Ser Asn Leu Arg Ala Leu Thr Ser Leu 835 840 845 Asp Ile Ser Asn Asn Val Glu Ala Thr Ser Leu Pro Glu Glu Met Phe 850 855 860 Lys Ser Leu Ala Asn Leu Lys Tyr Leu Asn Ile Ser Phe Phe Arg Asn 865 870 875 880 Leu Lys Glu Leu Pro Thr Ser Leu Ala Ser Leu Asn Ala Leu Lys Ser 885 890 895 Leu Lys Phe Glu Phe Cys Asn Ala Leu Glu Ser Leu Pro Ala Glu Gly 900 905 910 Val Lys Gly Leu Thr Ser Leu Thr Glu Leu Ser Val Ser Asn Cys Met 915 920 925 Met Leu Lys Cys Leu Pro Glu Gly Leu Gln His Leu Thr Ala Leu Thr 930 935 940 Thr Leu Thr Ile Thr Gln Cys Pro Ile Val Phe Lys Arg Cys Glu Arg 945 950 955 960 Gly Ile Gly Glu Asp Trp His Lys Ile Ala His Ile Pro Tyr Leu Thr 965 970 975 Leu Tyr Glu 56 992 PRT Artificial Sequence Description of Artificial Sequence alignment RGC1-blb 56 Met Ala Glu Ala Phe Leu Gln Val Leu Leu Asp Asn Leu Thr Phe Phe 1 5 10 15 Ile Gln Gly Glu Leu Gly Leu Val Phe Gly Phe Glu Lys Glu Phe Lys 20 25 30 Lys Leu Ser Ser Met Phe Ser Met Ile Gln Ala Val Leu Glu Asp Ala 35 40 45 Gln Glu Lys Gln Leu Lys Tyr Lys Ala Ile Lys Asn Trp Leu Gln Lys 50 55 60 Leu Asn Val Ala Ala Tyr Glu Val Asp Asp Ile Leu Asp Asp Cys Lys 65 70 75 80 Thr Glu Ala Ala Arg Phe Lys Gln Ala Val Leu Gly Arg Tyr His Pro 85 90 95 Arg Thr Ile Thr Phe Cys Tyr Lys Val Gly Lys Arg Met Lys Glu Met 100 105 110 Met Glu Lys Leu Asp Ala Ile Ala Glu Glu Arg Arg Asn Phe His Leu 115 120 125 Asp Glu Arg Ile Ile Glu Arg Gln Ala Ala Arg Arg Gln Thr Gly Phe 130 135 140 Val Leu Thr Glu Pro Lys Val Tyr Gly Arg Glu Lys Glu Glu Asp Glu 145 150 155 160 Ile Val Lys Ile Leu Ile Asn Asn Val Ser Tyr Ser Glu Glu Val Pro 165 170 175 Val Leu Pro Ile Leu Gly Met Gly Gly Leu Gly Lys Thr Thr Leu Ala 180 185 190 Gln Met Val Phe Asn Asp Gln Arg Ile Thr Glu His Phe Asn Leu Lys 195 200 205 Ile Trp Val Cys Val Ser Asp Asp Phe Asp Glu Lys Arg Leu Ile Lys 210 215 220 Ala Ile Val Glu Ser Ile Glu Gly Lys Ser Leu Gly Asp Met Asp Leu 225 230 235 240 Ala Pro Leu Gln Lys Lys Leu Gln Glu Leu Leu Asn Gly Lys Arg Tyr 245 250 255 Phe Leu Val Leu Asp Asp Val Trp Asn Glu Asp Gln Glu Lys Trp Asp 260 265 270 Asn Leu Arg Ala Val Leu Lys Ile Gly Ala Ser Gly Ala Ser Ile Leu 275 280 285 Ile Thr Thr Arg Leu Glu Lys Ile Gly Ser Ile Met Gly Thr Leu Gln 290 295 300 Leu Tyr Gln Leu Ser Asn Leu Ser Gln Glu Asp Cys Trp Leu Leu Phe 305 310 315 320 Lys Gln Arg Ala Phe Cys His Gln Thr Glu Thr Ser Pro Lys Leu Met 325 330 335 Glu Ile Gly Lys Glu Ile Val Lys Lys Cys Gly Gly Val Pro Leu Ala 340 345 350 Ala Lys Thr Leu Gly Gly Leu Leu Arg Phe Lys Arg Glu Glu Ser Glu 355 360 365 Trp Glu His Val Arg Asp Ser Glu Ile Trp Asn Leu Pro Gln Asp Glu 370 375 380 Asn Ser Val Leu Pro Ala Leu Arg Leu Ser Tyr His His Leu Pro Leu 385 390 395 400 Asp Leu Arg Gln Cys Phe Ala Tyr Cys Ala Val Phe Pro Lys Asp Thr 405 410 415 Lys Ile Glu Lys Glu Tyr Leu Ile Ala Leu Trp Met Ala His Ser Phe 420 425 430 Leu Leu Ser Lys Gly Asn Met Glu Leu Glu Asp Val Gly Asn Glu Val 435 440 445 Trp Asn Glu Leu Tyr Leu Arg Ser Phe Phe Gln Glu Ile Glu Val Lys 450 455 460 Ser Gly Lys Thr Tyr Phe Lys Met His Asp Leu Ile His Asp Leu Ala 465 470 475 480 Thr Ser Met Phe Ser Ala Ser Ala Ser Ser Arg Ser Ile Arg Gln Ile 485 490 495 Asn Val Lys Asp Asp Glu Asp Met Met Phe Ile Val Thr Asn Tyr Lys 500 505 510 Asp Met Met Ser Ile Gly Phe Ser Glu Val Val Ser Ser Tyr Ser Pro 515 520 525 Ser Leu Phe Lys Arg Phe Val Ser Leu Arg Val Leu Asn Leu Ser Asn 530 535 540 Ser Glu Phe Glu Gln Leu Pro Ser Ser Val Gly Asp Leu Val His Leu 545 550 555 560 Arg Tyr Leu Asp Leu Ser Gly Asn Lys Ile Cys Ser Leu Pro Lys Arg 565 570 575 Leu Cys Lys Leu Gln Asn Leu Gln Thr Leu Asp Leu Tyr Asn Cys Gln 580 585 590 Ser Leu Ser Cys Leu Pro Lys Gln Thr Ser Lys Leu Cys Ser Leu Arg 595 600 605 Asn Leu Val Leu Asp His Cys Pro Leu Thr Ser Met Pro Pro Arg Ile 610 615 620 Gly Leu Leu Thr Cys Leu Lys Thr Leu Gly Tyr Phe Val Val Gly Glu 625 630 635 640 Arg Lys Gly Tyr Gln Leu Gly Glu Leu Arg Asn Leu Asn Leu Arg Gly 645 650 655 Ala Ile Ser Ile Thr His Leu Glu Arg Val Lys Asn Asp Met Glu Ala 660 665 670 Lys Glu Ala Asn Leu Ser Ala Lys Ala Asn Leu His Ser Leu Ser Met 675 680 685 Ser Trp Asp Arg Pro Asn Arg Tyr Glu Ser Glu Glu Val Lys Val Leu 690 695 700 Glu Ala Leu Lys Pro His Pro Asn Leu Lys Tyr Leu Glu Ile Ile Asp 705 710 715 720 Phe Cys Gly Phe Cys Leu Pro Asp Trp Met Asn His Ser Val Leu Lys 725 730 735 Asn Val Val Ser Ile Leu Ile Ser Gly Cys Glu Asn Cys Ser Cys Leu 740 745 750 Pro Pro Phe Gly Glu Leu Pro Cys Leu Glu Ser Leu Glu Leu Gln Asp 755 760 765 Gly Ser Val Glu Val Glu Tyr Val Glu Asp Ser Gly Phe Leu Thr Arg 770 775 780 Arg Arg Phe Pro Ser Leu Arg Lys Leu His Ile Gly Gly Phe Cys Asn 785 790 795 800 Leu Lys Gly Leu Gln Arg Met Lys Gly Ala Glu Gln Phe Pro Val Leu 805 810 815 Glu Glu Met Lys Ile Ser Asp Cys Pro Met Phe Val Phe Pro Thr Leu 820 825 830 Ser Ser Val Lys Lys Leu Glu Ile Trp Gly Glu Ala Asp Ala Gly Gly 835 840 845 Leu Ser Ser Ile Ser Asn Leu Ser Thr Leu Thr Ser Leu Lys Ile Phe 850 855 860 Ser Asn His Thr Val Thr Ser Leu Leu Glu Glu Met Phe Lys Asn Leu 865 870 875 880 Glu Asn Leu Ile Tyr Leu Ser Val Ser Phe Leu Glu Asn Leu Lys Glu 885 890 895 Leu Pro Thr Ser Leu Ala Ser Leu Asn Asn Leu Lys Cys Leu Asp Ile 900 905 910 Arg Tyr Cys Tyr Ala Leu Glu Ser Leu Pro Glu Glu Gly Leu Glu Gly 915 920 925 Leu Ser Ser Leu Thr Glu Leu Phe Val Glu His Cys Asn Met Leu Lys 930 935 940 Cys Leu Pro Glu Gly Leu Gln His Leu Thr Thr Leu Thr Ser Leu Lys 945 950 955 960 Ile Arg Gly Cys Pro Gln Leu Ile Lys Arg Cys Glu Lys Gly Ile Gly 965 970 975 Glu Asp Trp His Lys Ile Ser His Ile Pro Asn Val Asn Ile Tyr Ile 980 985 990 57 1040 PRT Artificial Sequence Description of Artificial Sequence alignment RGC4-blb/RGA4-blb 57 Met Ala Glu Ala Phe Leu Gln Val Leu Leu Glu Asn Leu Thr Ser Phe 1 5 10 15 Ile Gly Asp Lys Leu Val Leu Ile Phe Gly Phe Glu Lys Glu Cys Glu 20 25 30 Lys Leu Ser Ser Val Phe Ser Thr Ile Gln Ala Val Leu Gln Asp Ala 35 40 45 Gln Glu Lys Gln Leu Lys Asp Lys Ala Ile Glu Asn Trp Leu Gln Lys 50 55 60 Leu Asn Ser Ala Ala Tyr Glu Val Asp Asp Ile Leu Gly Glu Cys Lys 65 70 75 80 Asn Glu Ala Ile Arg Phe Glu Gln Ser Arg Leu Gly Phe Tyr His Pro 85 90 95 Gly Ile Ile Asn Phe Arg His Lys Ile Gly Arg Arg Met Lys Glu Ile 100 105 110 Met Glu Lys Leu Asp Ala Ile Ser Glu Glu Arg Arg Lys Phe His Phe 115 120 125 Leu Glu Lys Ile Thr Glu Arg Gln Ala Ala Ala Ala Thr Arg Glu Thr 130 135 140 Val Gly Trp Gln Trp Gly Trp Ala Arg Leu Glu Tyr Lys Arg Leu Leu 145 150 155 160 Leu Gly Val Leu Met Arg Ile Met Ser Leu Arg Met His Val Ser Thr 165 170 175 Cys Ser Thr Leu Tyr Glu Phe Lys Phe Tyr Leu Cys Thr Pro Lys Val 180 185 190 Gly Ala Arg Arg Cys Phe Val Leu Thr Glu Pro Lys Val Tyr Gly Arg 195 200 205 Asp Lys Glu Glu Asp Glu Ile Val Lys Ile Leu Ile Asn Asn Val Asn 210 215 220 Val Ala Glu Glu Leu Pro Val Phe Pro Ile Ile Gly Met Gly Gly Leu 225 230 235 240 Gly Lys Thr Thr Leu Ala Gln Met Ile Phe Asn Asp Glu Arg Val Thr 245 250 255 Lys His Phe Asn Pro Lys Ile Trp Val Cys Val Ser Asp Asp Phe Asp 260 265 270 Glu Lys Arg Leu Ile Lys Thr Ile Ile Gly Asn Ile Glu Arg Ser Ser 275 280 285 Pro His Val Glu Asp Leu Ala Ser Phe Gln Lys Lys Leu Gln Glu Leu 290 295 300 Leu Asn Gly Lys Arg Tyr Leu Leu Val Leu Asp Asp Val Trp Asn Asp 305 310 315 320 Asp Leu Glu Lys Trp Ala Lys Leu Arg Ala Val Leu Thr Val Gly Ala 325 330 335 Arg Gly Ala Ser Ile Leu Ala Thr Thr Arg Leu Glu Lys Val Gly Ser 340 345 350 Ile Met Gly Thr Leu Gln Pro Tyr His Leu Ser Asn Leu Ser Pro His 355 360 365 Asp Ser Leu Leu Leu Phe Met Gln Arg Ala Phe Gly Gln Gln Lys Glu 370 375 380 Ala Asn Pro Asn Leu Val Ala Ile Gly Lys Glu Ile Val Lys Lys Cys 385 390 395 400 Gly Gly Val Pro Leu Ala Ala Lys Thr Leu Gly Gly Leu Leu Arg Phe 405 410 415 Lys Arg Glu Glu Ser Glu Trp Glu His Val Arg Asp Asn Glu Ile Trp 420 425 430 Ser Leu Pro Gln Asp Glu Ser Ser Ile Leu Pro Ala Leu Arg Leu Ser 435 440 445 Tyr His His Leu Pro Leu Asp Leu Arg Gln Cys Phe Ala Tyr Cys Ala 450 455 460 Val Phe Pro Lys Asp Thr Lys Met Ile Lys Glu Asn Leu Ile Thr Leu 465 470 475 480 Trp Met Ala His Gly Phe Leu Leu Ser Lys Gly Asn Leu Glu Leu Glu 485 490 495 Asp Val Gly Asn Glu Val Trp Asn Glu Leu Tyr Leu Arg Ser Phe Phe 500 505 510 Gln Glu Ile Glu Ala Lys Ser Gly Asn Thr Tyr Phe Lys Ile His Asp 515 520 525 Leu Ile His Asp Leu Ala Thr Ser Leu Phe Ser Ala Ser Ala Ser Cys 530 535 540 Gly Asn Ile Arg Glu Ile Asn Val Lys Asp Tyr Lys His Thr Val Ser 545 550 555 560 Ile Gly Phe Ala Ala Val Val Ser Ser Tyr Ser Pro Ser Leu Leu Lys 565 570 575 Lys Phe Val Ser Leu Arg Val Leu Asn Leu Ser Tyr Ser Lys Leu Glu 580 585 590 Gln Leu Pro Ser Ser Ile Gly Asp Leu Leu His Leu Arg Tyr Leu Asp 595 600 605 Leu Ser Cys Asn Asn Phe Arg Ser Leu Pro Glu Arg Leu Cys Lys Leu 610 615 620 Gln Asn Leu Gln Thr Leu Asp Val His Asn Cys Tyr Ser Leu Asn Cys 625 630 635 640 Leu Pro Lys Gln Thr Ser Lys Leu Ser Ser Leu Arg His Leu Val Val 645 650 655 Asp Gly Cys Pro Leu Thr Ser Thr Pro Pro Arg Ile Gly Leu Leu Thr 660 665 670 Cys Leu Lys Thr Leu Gly Phe Phe Ile Val Gly Ser Lys Lys Gly Tyr 675 680 685 Gln Leu Gly Glu Leu Lys Asn Leu Asn Leu Cys Gly Ser Ile Ser Ile 690 695 700 Thr His Leu Glu Arg Val Lys Asn Asp Thr Asp Ala Glu Ala Asn Leu 705 710 715 720 Ser Ala Lys Ala Asn Leu Gln Ser Leu Ser Met Ser Trp Asp Asn Asp 725 730 735 Gly Pro Asn Arg Tyr Glu Ser Lys Glu Val Lys Val Leu Glu Ala Leu 740 745 750 Lys Pro His Pro Asn Leu Lys Tyr Leu Glu Ile Ile Ala Phe Gly Gly 755 760 765 Phe Arg Phe Pro Ser Trp Ile Asn His Ser Val Leu Glu Lys Val Ile 770 775 780 Ser Val Arg Ile Lys Ser Cys Lys Asn Cys Leu Cys Leu Pro Pro Phe 785 790 795 800 Gly Glu Leu Pro Cys Leu Glu Asn Leu Glu Leu Gln Asn Gly Ser Ala 805 810 815 Glu Val Glu Tyr Val Glu Glu Asp Asp Val His Ser Arg Phe Ser Thr 820 825 830 Arg Arg Ser Phe Pro Ser Leu Lys Lys Leu Arg Ile Trp Phe Phe Arg 835 840 845 Ser Leu Lys Gly Leu Met Lys Glu Glu Gly Glu Glu Lys Phe Pro Met 850 855 860 Leu Glu Glu Met Ala Ile Leu Tyr Cys Pro Leu Phe Val Phe Pro Thr 865 870 875 880 Leu Ser Ser Val Lys Lys Leu Glu Val His Gly Asn Thr Asn Thr Arg 885 890 895 Gly Leu Ser Ser Ile Ser Asn Leu Ser Thr Leu Thr Ser Leu Arg Ile 900 905 910 Gly Ala Asn Tyr Arg Ala Thr Ser Leu Pro Glu Glu Met Phe Thr Ser 915 920 925 Leu Thr Asn Leu Glu Phe Leu Ser Phe Phe Asp Phe Lys Asn Leu Lys 930 935 940 Asp Leu Pro Thr Ser Leu Thr Ser Leu Asn Ala Leu Lys Arg Leu Gln 945 950 955 960 Ile Glu Ser Cys Asp Ser Leu Glu Ser Phe Pro Glu Gln Gly Leu Glu 965 970 975 Gly Leu Thr Ser Leu Thr Gln Leu Phe Val Lys Tyr Cys Lys Met Leu 980 985 990 Lys Cys Leu Pro Glu Gly Leu Gln His Leu Thr Ala Leu Thr Asn Leu 995 1000 1005 Gly Val Ser Gly Cys Pro Glu Val Glu Lys Arg Cys Asp Lys Glu Ile 1010 1015 1020 Gly Glu Asp Trp His Lys Ile Ala His Ile Pro Asn Leu Asp Ile His 1025 1030 1035 1040 58 979 PRT Artificial Sequence Description of Artificial Sequence alignment RGA3-blb 58 Met Ala Glu Ala Phe Ile Gln Val Val Leu Asp Asn Leu Thr Ser Phe 1 5 10 15 Leu Lys Gly Glu Leu Val Leu Leu Phe Gly Phe Gln Asp Glu Phe Gln 20 25 30 Arg Leu Ser Ser Met Phe Ser Thr Ile Gln Ala Val Leu Glu Asp Ala 35 40 45 Gln Glu Lys Gln Leu Asn Asp Lys Pro Leu Glu Asn Trp Leu Gln Lys 50 55 60 Leu Asn Ala Ala Thr Tyr Glu Val Asp Asp Ile Leu Asp Glu Tyr Lys 65 70 75 80 Thr Lys Ala Thr Arg Phe Leu Gln Ser Glu Tyr Gly Arg Tyr His Pro 85 90 95 Lys Val Ile Pro Phe Arg His Lys Val Gly Lys Arg Met Asp Gln Val 100 105 110 Met Lys Lys Leu Asn Ala Ile Ala Glu Glu Arg Lys Asn Phe His Leu 115 120 125 Gln Glu Lys Ile Ile Glu Arg Gln Ala Ala Thr Arg Glu Thr Gly Ser 130 135 140 Val Leu Thr Glu Pro Gln Val Tyr Gly Arg Asp Lys Glu Lys Asp Glu 145 150 155 160 Ile Val Lys Ile Leu Ile Asn Asn Val Ser Asp Ala Gln Lys Leu Ser 165 170 175 Val Leu Pro Ile Leu Gly Met Gly Gly Leu Gly Lys Thr Thr Leu Ser 180 185 190 Gln Met Val Phe Asn Asp Gln Arg Val Thr Glu Arg Phe Tyr Pro Lys 195 200 205 Ile Trp Ile Cys Val Ser Asp Asp Phe Asp Glu Lys Arg Leu Ile Lys 210 215 220 Ala Ile Val Glu Ser Ile Glu Gly Lys Ser Leu Ser Asp Met Asp Leu 225 230 235 240 Ala Pro Leu Gln Lys Lys Leu Gln Glu Leu Leu Asn Gly Lys Arg Tyr 245 250 255 Phe Leu Val Leu Asp Asp Val Trp Asn Glu Asp Gln His Lys Trp Ala 260 265 270 Asn Leu Arg Ala Val Leu Lys Val Gly Ala Ser Gly Ala Phe Val Leu 275 280 285 Thr Thr Thr Arg Leu Glu Lys Val Gly Ser Ile Met Gly Thr Leu Gln 290 295 300 Pro Tyr Glu Leu Ser Asn Leu Ser Pro Glu Asp Cys Trp Phe Leu Phe 305 310 315 320 Met Gln Arg Ala Phe Gly His Gln Glu Glu Ile Asn Pro Asn Leu Val 325 330 335 Ala Ile Gly Lys Glu Ile Val Lys Lys Cys Gly Gly Val Pro Leu Ala 340 345 350 Ala Lys Thr Leu Gly Gly Ile Leu Arg Phe Lys Arg Glu Glu Arg Ala 355 360 365 Trp Glu His Val Arg Asp Ser Pro Ile Trp Asn Leu Pro Gln Asp Glu 370 375 380 Ser Ser Ile Leu Pro Ala Leu Arg Leu Ser Tyr His His Leu Pro Leu 385 390 395 400 Asp Leu Asp Gln Cys Phe Val Tyr Cys Ala Val Phe Pro Lys Asp Thr 405 410 415 Lys Met Ala Lys Glu Asn Leu Ile Ala Phe Trp Met Ala His Gly Phe 420 425 430 Leu Leu Ser Lys Gly Asn Leu Glu Leu Glu Asp Val Gly Asp Glu Val 435 440 445 Trp Asn Glu Leu Tyr Leu Arg Ser Phe Phe Gln Glu Ile Glu Val Glu 450 455 460 Ser Gly Lys Thr Tyr Phe Lys Met His Asp Leu Ile His Asp Leu Ala 465 470 475 480 Thr Ser Leu Phe Ser Ala Asn Thr Ser Ser Ser Asn Ile Arg Glu Ile 485 490 495 Asn Ala Asn Tyr Asp Gly Tyr Met Met Ser Ile Gly Phe Ala Glu Val 500 505 510 Val Ser Ser Tyr Ser Pro Ser Leu Leu Gln Lys Phe Val Ser Leu Arg 515 520 525 Val Leu Asn Leu Arg Asn Ser Asn Leu Asn Gln Leu Pro Ser Ser Ile 530 535 540 Gly Asp Leu Val His Leu Arg Tyr Leu Asp Leu Ser Gly Asn Phe Arg 545 550 555 560 Ile Arg Asn Leu Pro Lys Arg Leu Cys Arg Leu Gln Asn Leu Gln Thr 565 570 575 Leu Asp Leu His Tyr Cys Asp Ser Leu Ser Cys Leu Pro Lys Gln Thr 580 585 590 Ser Lys Leu Gly Ser Leu Arg Asn Leu Leu Leu Asp Gly Cys Ser Leu 595 600 605 Thr Ser Thr Pro Pro Arg Ile Gly Leu Leu Thr Cys Leu Lys Ser Leu 610 615 620 Ser Cys Phe Val Ile Gly Lys Arg Lys Gly Tyr Gln Leu Gly Glu Leu 625 630 635 640 Lys Asn Leu Asn Leu Tyr Gly Ser Ile Ser Ile Thr Lys Leu Asp Arg 645 650 655 Val Lys Lys Asp Ser Asp Ala Lys Glu Ala Asn Leu Ser Ala Lys Ala 660 665 670 Asn Leu His Ser Leu Cys Leu Ser Trp Asp Leu Asp Gly Lys His Arg 675 680 685 Tyr Asp Ser Glu Val Leu Glu Ala Leu Lys Pro His Ser Asn Leu Lys 690 695 700 Tyr Leu Glu Ile Asn Gly Phe Gly Gly Ile Arg Leu Pro Asp Trp Met 705 710 715 720 Asn Gln Ser Val Leu Lys Asn Val Val Ser Ile Arg Ile Arg Gly Cys 725 730 735 Glu Asn Cys Ser Cys Leu Pro Pro Phe Gly Glu Leu Pro Cys Leu Glu 740 745 750 Ser Leu Glu Leu His Thr Gly Ser Ala Asp Val Glu Tyr Val Glu Asp 755 760 765 Asn Val His Pro Gly Arg Phe Pro Ser Leu Arg Lys Leu Val Ile Trp 770 775 780 Asp Phe Ser Asn Leu Lys Gly Leu Leu Lys Lys Glu Gly Glu Glu Gln 785 790 795 800 Phe Pro Val Leu Glu Glu Met Thr Phe Tyr Trp Cys Pro Met Phe Val 805 810 815 Ile Pro Thr Leu Ser Ser Val Lys Thr Leu Lys Val Ile Ala Thr Asp 820 825 830 Ala Thr Val Leu Arg Ser Ile Ser Asn Leu Arg Ala Leu Thr Ser Leu 835 840 845 Asp Ile Ser Asn Asn Val Glu Ala Thr Ser Leu Pro Glu Glu Met Phe 850 855 860 Lys Ser Leu Ala Asn Leu Lys Tyr Leu Asn Ile Ser Phe Phe Arg Asn 865 870 875 880 Leu Lys Glu Leu Pro Thr Ser Leu Ala Ser Leu Asn Ala Leu Lys Ser 885 890 895 Leu Lys Phe Glu Phe Cys Asn Ala Leu Glu Ser Leu Pro Ala Glu Gly 900 905 910 Val Lys Gly Leu Thr Ser Leu Thr Glu Leu Ser Val Ser Asn Cys Met 915 920 925 Met Leu Lys Cys Leu Pro Glu Gly Leu Gln His Leu Thr Ala Leu Thr 930 935 940 Thr Leu Thr Ile Thr Gln Cys Pro Ile Val Phe Lys Arg Cys Glu Arg 945 950 955 960 Gly Ile Gly Glu Asp Trp His Lys Ile Ala His Ile Pro Tyr Leu Thr 965 970 975 Leu Tyr Glu 59 945 PRT Artificial Sequence Description of Artificial Sequence alignment SH10-tub 59 Met Ala Glu Ala Phe Ile Gln Val Leu Ile Asp Asn Leu Thr Ser Phe 1 5 10 15 Leu Lys Gly Glu Leu Val Leu Leu Phe Gly Phe Gln Asn Glu Phe Gln 20 25 30 Arg Leu Ser Ser Ile Phe Ser Thr Ile Gln Ala Val Leu Glu Asp Ala 35 40 45 Gln Glu Lys Gln Leu Asn Asp Lys Pro Leu Glu Asn Trp Leu Gln Lys 50 55 60 Leu Asn Ala Ala Thr Tyr Glu Val Asp Asp Ile Leu Asp Glu Tyr Lys 65 70 75 80 Thr Lys Ala Thr Arg Phe Ser Gln Ser Ala Tyr Gly Arg Tyr His Pro 85 90 95 Lys Val Ile Pro Phe Arg His Lys Val Gly Lys Arg Met Asp Gln Val 100 105 110 Met Lys Lys Leu Asn Ala Ile Ala Glu Glu Arg Lys Asn Phe His Leu 115 120 125 His Glu Lys Ile Ile Glu Arg Gln Ala Val Arg Arg Glu Thr Gly Ser 130 135 140 Val Leu Thr Glu Pro Gln Val Tyr Gly Arg Asp Lys Glu Glu Asp Glu 145 150 155 160 Ile Val Lys Ile Leu Ile Asn Asn Val Ser Asp Ala Gln His Leu Ser 165 170 175 Val Leu Pro Ile Leu Gly Met Gly Gly Leu Gly Lys Thr Thr Leu Ala 180 185 190 Gln Met Val Phe Asn Asp Gln Arg Ile Thr Glu His Phe His Ser Lys 195 200 205 Ile Trp Ile Cys Val Ser Glu Asp Phe Asp Glu Lys Arg Leu Leu Lys 210 215 220 Ala Ile Ile Glu Ser Ile Glu Gly Arg Pro Leu Leu Gly Glu Met Asp 225 230 235 240 Leu Ala Pro Leu Gln Lys Lys Leu Gln Glu Leu Leu Asn Gly Lys Arg 245 250 255 Tyr Phe Leu Val Leu Asp Asp Val Trp Asn Glu Asp Gln Gln Lys Trp 260 265 270 Ala Asn Leu Arg Ala Val Leu Lys Val Gly Ala Ser Gly Ala Phe Val 275 280 285 Leu Ala Thr Thr Arg Leu Glu Lys Val Gly Ser Ile Met Gly Thr Leu 290 295 300 Gln Pro Tyr Glu Leu Ser Asn Leu Ser Gln Glu Asp Cys Trp Leu Leu 305 310 315 320 Phe Ile Gln Cys Ala Phe Gly His Gln Glu Glu Ile Asn Pro Asn Leu 325 330 335 Val Ala Ile Gly Lys Glu Ile Val Lys Lys Ser Gly Gly Val Pro Leu 340 345 350 Ala Ala Lys Thr Leu Gly Gly Ile Leu Arg Phe Lys Arg Glu Glu Arg 355 360 365 Ala Trp Glu His Val Arg Asp Ser Glu Ile Trp Asn Leu Pro Gln Glu 370 375 380 Glu Arg Ser Ile Leu Pro Ala Leu Arg Leu Ser Tyr His His Leu Pro 385 390 395 400 Leu Asp Leu Arg Gln Cys Phe Ala Tyr Cys Ala Val Phe Pro Lys Asp 405 410 415 Thr Lys Met Glu Lys Glu Lys Leu Ile Ser Leu Trp Met Ala His Gly 420 425 430 Phe Leu Leu Leu Glu Gly Lys Leu Gln Pro Glu Asp Val Gly Asn Glu 435 440 445 Val Ser Lys Glu Leu Cys Leu Arg Ser Phe Phe Gln Glu Ile Glu Ala 450 455 460 Lys Cys Gly Lys Thr Tyr Phe Lys Met His Asp Leu His His Asp Leu 465 470 475 480 Ala Thr Ser Leu Phe Ser Ala Ser Thr Ser Ser Ser Asn Ile Arg Glu 485 490 495 Ile Asn Val Lys Gly Tyr Pro His Lys Met Ser Ile Gly Phe Thr Glu 500 505 510 Val Val Ser Ser Tyr Ser Pro Ser Leu Ser Gln Lys Phe Val Ser Leu 515 520 525 Arg Val Leu Asn Leu Ser Asn Leu His Phe Glu Glu Leu Ser Ser Ser 530 535 540 Ile Gly Asp Leu Val His Met Arg Cys Leu Asp Leu Ser Glu Asn Ser 545 550 555 560 Gly Ile Arg Ser Leu Pro Lys Gln Leu Cys Lys Leu Gln Asn Leu Gln 565 570 575 Thr Leu Asp Leu His Asn Cys Tyr Ser Leu Ser Cys Leu Pro Lys Glu 580 585 590 Pro Ser Lys Leu Gly Ser Leu Arg Asn Leu Phe Phe His Gly Cys Asp 595 600 605 Glu Leu Asn Ser Met Pro Pro Arg Ile Gly Ser Leu Thr Phe Leu Lys 610 615 620 Thr Leu Lys Trp Ile Cys Cys Gly Ile Lys Lys Gly Tyr Gln Leu Gly 625 630 635 640 Lys Leu Arg Asp Val Asn Leu Tyr Gly Ser Ile Glu Ile Thr His Leu 645 650 655 Glu Arg Val Lys Asn Val Met Asp Ala Lys Glu Ala Asn Leu Ser Ala 660 665 670 Lys Gly Asn Leu His Ser Leu Ile Met Asn Trp Ser Arg Lys Gly Pro 675 680 685 His Ile Tyr Glu Ser Glu Glu Val Arg Val Ile Glu Ala Leu Lys Pro 690 695 700 His Pro Asn Leu Thr Cys Leu Thr Ile Ser Gly Phe Arg Gly Phe Arg 705 710 715 720 Phe Pro Glu Trp Met Asn His Ser Val Leu Lys Asn Val Val Ser Ile 725 730 735 Glu Ile Ser Gly Cys Lys Asn Cys Ser Cys Leu Pro Pro Phe Gly Glu 740 745 750 Leu Pro Cys Leu Lys Arg Leu Glu Leu Gln Lys Gly Ser Ala Glu Val 755 760 765 Glu Tyr Val Asp Ser Gly Phe Pro Thr Arg Arg Arg Phe Pro Ser Leu 770 775 780 Arg Lys Leu Phe Ile Gly Glu Phe Pro Asn Leu Lys Gly Leu Leu Lys 785 790 795 800 Lys Glu Gly Glu Glu Gln Phe Pro Val Leu Glu Arg Met Thr Ile Phe 805 810 815 Tyr Cys His Met Phe Val Tyr Thr Thr Leu Ser Asn Phe Arg Ala Leu 820 825 830 Thr Ser Leu His Ile Ser His Asn Asn Glu Ala Thr Ser Leu Pro Glu 835 840 845 Glu Ile Phe Lys Ser Phe Ala Asn Leu Lys Tyr Leu Lys Ile Ser Leu 850 855 860 Phe Tyr Asn Leu Lys Glu Leu Pro Ser Ser Leu Ala Cys Leu Asn Ala 865 870 875 880 Leu Lys Thr Leu Glu Ile His Ser Cys Ser Ala Leu Glu Ser Leu Pro 885 890 895 Glu Glu Gly Val Lys Gly Leu Thr Ser Leu Thr Glu Leu Phe Val Tyr 900 905 910 Asp Cys Glu Met Leu Lys Phe Leu Pro Glu Gly Leu Gln His Leu Thr 915 920 925 Ala Leu Thr Ser Leu Lys Leu Arg Arg Cys Pro Gln Leu Ile Lys Arg 930 935 940 Cys 945 60 992 PRT Artificial Sequence Description of Artificial Sequence alignment RGA1-blb 60 Met Ala Glu Ala Phe Leu Gln Val Leu Leu Asp Asn Leu Thr Phe Phe 1 5 10 15 Ile Gln Gly Glu Leu Gly Leu Val Phe Gly Phe Glu Lys Glu Phe Lys 20 25 30 Lys Leu Ser Ser Met Phe Ser Met Ile Gln Ala Val Leu Glu Asp Ala 35 40 45 Gln Glu Lys Gln Leu Lys Tyr Lys Ala Ile Lys Asn Trp Leu Gln Lys 50 55 60 Leu Asn Val Ala Ala Tyr Glu Val Asp Asp Ile Leu Asp Asp Cys Lys 65 70 75 80 Thr Glu Ala Ala Arg Phe Lys Gln Ala Val Leu Gly Arg Tyr His Pro 85 90 95 Arg Thr Ile Thr Phe Cys Tyr Lys Val Gly Lys Arg Met Lys Glu Met 100 105 110 Met Glu Lys Leu Asp Ala Ile Ala Glu Glu Arg Arg Asn Phe His Leu 115 120 125 Asp Glu Arg Ile Ile Glu Arg Gln Ala Ala Arg Arg Gln Thr Gly Phe 130 135 140 Val Leu Thr Glu Pro Lys Val Tyr Gly Arg Glu Lys Glu Glu Asp Glu 145 150 155 160 Ile Val Lys Ile Leu Ile Asn Asn Val Ser Tyr Ser Glu Glu Val Pro 165 170 175 Val Leu Pro Ile Leu Gly Met Gly Gly Leu Gly Lys Thr Thr Leu Ala 180 185 190 Gln Met Val Phe Asn Asp Gln Arg Ile Thr Glu His Phe Asn Leu Lys 195 200 205 Ile Trp Val Cys Val Ser Asp Asp Phe Asp Glu Lys Arg Leu Ile Lys 210 215 220 Ala Ile Val Glu Ser Ile Glu Gly Lys Ser Leu Gly Asp Met Asp Leu 225 230 235 240 Ala Pro Leu Gln Lys Lys Leu Gln Glu Leu Leu Asn Gly Lys Arg Tyr 245 250 255 Phe Leu Val Leu Asp Asp Val Trp Asn Glu Asp Gln Glu Lys Trp Asp 260 265 270 Asn Leu Arg Ala Val Leu Lys Ile Gly Ala Ser Gly Ala Ser Ile Leu 275 280 285 Ile Thr Thr Arg Leu Glu Lys Ile Gly Ser Ile Met Gly Thr Leu Gln 290 295 300 Leu Tyr Gln Leu Ser Asn Leu Ser Gln Glu Asp Cys Trp Leu Leu Phe 305 310 315 320 Lys Gln Arg Ala Phe Cys His Gln Thr Glu Thr Ser Pro Lys Leu Met 325 330 335 Glu Ile Gly Lys Glu Ile Val Lys Lys Cys Gly Gly Val Pro Leu Ala 340 345 350 Ala Lys Thr Leu Gly Gly Leu Leu Arg Phe Lys Arg Glu Glu Ser Glu 355 360 365 Trp Glu His Val Arg Asp Ser Glu Ile Trp Asn Leu Pro Gln Asp Glu 370 375 380 Asn Ser Val Leu Pro Ala Leu Arg Leu Ser Tyr His His Leu Pro Leu 385 390 395 400 Asp Leu Arg Gln Cys Phe Ala Tyr Cys Ala Val Phe Pro Lys Asp Thr 405 410 415 Lys Ile Glu Lys Glu Tyr Leu Ile Ala Leu Trp Met Ala His Ser Phe 420 425 430 Leu Leu Ser Lys Gly Asn Met Glu Leu Glu Asp Val Gly Asn Glu Val 435 440 445 Trp Asn Glu Leu Tyr Leu Arg Ser Phe Phe Gln Glu Ile Glu Val Lys 450 455 460 Ser Gly Lys Thr Tyr Phe Lys Met His Asp Leu Ile His Asp Leu Ala 465 470 475 480 Thr Ser Met Phe Ser Ala Ser Ala Ser Ser Arg Ser Ile Arg Gln Ile 485 490 495 Asn Val Lys Asp Asp Glu Asp Met Met Phe Ile Val Thr Asn Tyr Lys 500 505 510 Asp Met Met Ser Ile Gly Phe Ser Glu Val Val Ser Ser Tyr Ser Pro 515 520 525 Ser Leu Phe Lys Arg Phe Val Ser Leu Arg Val Leu Asn Leu Ser Asn 530 535 540 Ser Glu Phe Glu Gln Leu Pro Ser Ser Val Gly Asp Leu Val His Leu 545 550 555 560 Arg Tyr Leu Asp Leu Ser Gly Asn Lys Ile Cys Ser Leu Pro Lys Arg 565 570 575 Leu Cys Lys Leu Gln Asn Leu Gln Thr Leu Asp Leu Tyr Asn Cys Gln 580 585 590 Ser Leu Ser Cys Leu Pro Lys Gln Thr Ser Lys Leu Cys Ser Leu Arg 595 600 605 Asn Leu Val Leu Asp His Cys Pro Leu Thr Ser Met Pro Pro Arg Ile 610 615 620 Gly Leu Leu Thr Cys Leu Lys Thr Leu Gly Tyr Phe Val Val Gly Glu 625 630 635 640 Arg Lys Gly Tyr Gln Leu Gly Glu Leu Arg Asn Leu Asn Leu Arg Gly 645 650 655 Ala Ile Ser Ile Thr His Leu Glu Arg Val Lys Asn Asp Met Glu Ala 660 665 670 Lys Glu Ala Asn Leu Ser Ala Lys Ala Asn Leu His Ser Leu Ser Met 675 680 685 Ser Trp Asp Arg Pro His Arg Tyr Glu Ser Glu Glu Val Lys Val Leu 690 695 700 Glu Ala Leu Lys Pro His Pro Asn Leu Lys Tyr Leu Glu Ile Ile Asp 705 710 715 720 Phe Cys Gly Phe Cys Leu Pro Asp Trp Met Asn His Ser Val Leu Lys 725 730 735 Asn Val Val Ser Ile Leu Ile Ser Gly Cys Glu Asn Cys Ser Cys Leu 740 745 750 Pro Pro Phe Gly Glu Leu Pro Cys Leu Glu Ser Leu Glu Leu Gln Asp 755 760 765 Gly Ser Val Glu Val Glu Tyr Val Glu Asp Ser Gly Phe Leu Thr Arg 770 775 780 Arg Arg Phe Pro Ser Leu Arg Lys Leu His Ile Gly Gly Phe Cys Asn 785 790 795 800 Leu Lys Gly Leu Gln Arg Met Lys Gly Glu Glu Gln Phe Pro Val Leu 805 810 815 Glu Glu Met Lys Ile Ser Asp Cys Pro Met Phe Val Phe Pro Thr Leu 820 825 830 Ser Ser Val Lys Lys Leu Glu Ile Trp Gly Glu Ala Asp Ala Gly Gly 835 840 845 Leu Ser Ser Ile Ser Asn Leu Ser Thr Leu Thr Ser Leu Lys Ile Phe 850 855 860 Ser Asn His Thr Val Thr Ser Leu Leu Glu Glu Met Phe Lys Asn Leu 865 870 875 880 Glu Asn Leu Ile Tyr Leu Ser Val Ser Phe Leu Glu Asn Leu Lys Glu 885 890 895 Leu Pro Thr Ser Leu Ala Ser Leu Asn Asn Leu Lys Cys Leu Asp Ile 900 905 910 Arg Tyr Cys Tyr Ala Leu Glu Ser Leu Pro Glu Glu Gly Leu Glu Gly 915 920 925 Leu Ser Ser Leu Thr Glu Leu Phe Val Glu His Cys Asn Met Leu Lys 930 935 940 Cys Leu Pro Glu Gly Leu Gln His Leu Thr Thr Leu Thr Ser Leu Lys 945 950 955 960 Ile Arg Gly Cys Pro Gln Leu Ile Lys Arg Cys Glu Lys Gly Ile Gly 965 970 975 Glu Asp Trp His Lys Ile Ser His Ile Pro Asn Val Asn Ile Tyr Ile 980 985 990 61 972 PRT Artificial Sequence Description of Artificial Sequence alignment B149-blb 61 Met Ala Glu Ala Phe Ile Gln Val Leu Leu Asp Asn Leu Thr Phe Phe 1 5 10 15 Ile Gln Gly Glu Leu Gly Leu Val Phe Gly Phe Glu Lys Glu Phe Lys 20 25 30 Lys Leu Ser Ser Met Phe Ser Met Ile Gln Ala Val Leu Glu Asp Ala 35 40 45 Gln Glu Lys Gln Leu Lys Tyr Lys Ala Ile Lys Asn Trp Leu Gln Lys 50 55 60 Leu Asn Val Ala Ala Tyr Glu Val Asp Asp Ile Leu Asp Asp Cys Lys 65 70 75 80 Thr Glu Ala Ala Arg Phe Lys Gln Ala Val Leu Gly Arg Tyr His Pro 85 90 95 Arg Thr Ile Thr Phe Cys Tyr Lys Val Gly Lys Arg Met Lys Glu Met 100 105 110 Met Glu Lys Leu Asp Ala Ile Ala Glu Glu Arg Arg Asn Phe His Leu 115 120 125 Asp Glu Arg Ile Ile Glu Arg Gln Ala Ala Arg Arg Gln Thr Gly Phe 130 135 140 Val Leu Thr Glu Pro Lys Val Tyr Gly Arg Glu Lys Glu Glu Asp Glu 145 150 155 160 Ile Val Lys Ile Leu Ile Asn Asn Val Ser Tyr Ser Glu Glu Val Pro 165 170 175 Val Leu Pro Ile Leu Gly Met Gly Gly Leu Gly Lys Thr Thr Leu Ala 180 185 190 Gln Met Val Phe Asn Asp Gln Arg Ile Thr Glu His Phe Asn Leu Lys 195 200 205 Ile Trp Val Cys Val Ser Asp Asp Phe Asp Glu Lys Arg Leu Ile Lys 210 215 220 Ala Ile Val Glu Ser Ile Glu Gly Lys Ser Leu Gly Asp Met Asp Leu 225 230 235 240 Ala Pro Leu Gln Lys Lys Leu Gln Glu Leu Leu Asn Gly Lys Arg Tyr 245 250 255 Phe Leu Val Leu Asp Asp Val Trp Asn Glu Asp Gln Glu Lys Trp Asp 260 265 270 Asn Leu Arg Ala Val Leu Lys Ile Gly Ala Ser Gly Ala Ser Ile Leu 275 280 285 Ile Thr Thr Arg Leu Glu Lys Ile Gly Ser Ile Met Gly Thr Leu Gln 290 295 300 Leu Tyr Gln Leu Ser Asn Leu Ser Gln Glu Asp Cys Trp Leu Leu Phe 305 310 315 320 Lys Gln Arg Ala Phe Cys His Gln Thr Glu Thr Ser Pro Lys Leu Met 325 330 335 Glu Ile Gly Lys Glu Ile Val Lys Lys Cys Gly Gly Val Pro Leu Ala 340 345 350 Ala Lys Thr Leu Gly Gly Leu Leu Arg Phe Lys Arg Glu Glu Ser Glu 355 360 365 Trp Glu His Val Arg Asp Ser Glu Ile Trp Asn Leu Pro Gln Asp Glu 370 375 380 Asn Ser Val Leu Pro Ala Leu Arg Leu Ser Tyr His His Leu Pro Leu 385 390 395 400 Asp Leu Arg Gln Cys Phe Ala Tyr Cys Ala Val Phe Pro Lys Asp Thr 405 410 415 Lys Ile Glu Lys Glu Tyr Leu Ile Ala Leu Trp Met Ala His Ser Phe 420 425 430 Leu Leu Ser Lys Gly Asn Met Glu Leu Glu Asp Val Gly Asn Glu Val 435 440 445 Trp Asn Glu Leu Tyr Leu Arg Ser Phe Phe Gln Gly Ile Glu Val Lys 450 455 460 Ser Gly Lys Thr Tyr Phe Lys Met His Asp Leu Ile His Asp Leu Ala 465 470 475 480 Thr Ser Met Phe Ser Ala Ser Ala Ser Ser Arg Ser Ile Arg Gln Ile 485 490 495 Asn Val Lys Asp Asp Glu Asp Met Met Phe Ile Val Thr Asn Tyr Lys 500 505 510 Asp Met Met Ser Ile Gly Phe Ser Glu Val Val Ser Ser Tyr Ser Pro 515 520 525 Ser Leu Phe Lys Arg Phe Val Ser Leu Arg Val Leu Asn Leu Ser Asn 530 535 540 Ser Glu Phe Glu Gln Leu Pro Ser Ser Val Gly Asp Leu Val His Leu 545 550 555 560 Arg Tyr Leu Asp Leu Ser Gly Asn Lys Ile Cys Ser Leu Pro Lys Arg 565 570 575 Leu Cys Lys Leu Arg Asn Leu Gln Thr Leu Asp Leu Tyr Asn Cys Gln 580 585 590 Ser Leu Ser Cys Leu Pro Lys Gln Thr Ser Lys Leu Cys Ser Leu Arg 595 600 605 Asn Leu Val Leu Asp His Ser Cys Pro Leu Thr Ser Met Pro Pro Arg 610 615 620 Ile Gly Leu Leu Thr Cys Leu Lys Thr Leu Gly Tyr Phe Val Val Gly 625 630 635 640 Glu Arg Lys Gly Tyr Gln Leu Gly Glu Leu Arg Asn Leu Asn Leu Arg 645 650 655 Gly Ala Ile Ser Ile Thr His Leu Glu Arg Val Lys Asn Asp Met Glu 660 665 670 Ala Lys Glu Ala Asn Leu Ser Ala Lys Ala Asn Leu His Ser Leu Ser 675 680 685 Met Ser Trp Asp Arg Pro Asn Arg Tyr Glu Ser Glu Glu Val Lys Val 690 695 700 Leu Glu Ala Leu Lys Pro His Pro Asn Leu Lys Tyr Leu Glu Ile Ile 705 710 715 720 Asp Phe Cys Gly Phe Cys Leu Pro Asp Trp Met Asn His Ser Val Leu 725 730 735 Lys Asn Val Val Ser Ile Leu Ile Ser Gly Cys Glu Asn Cys Ser Cys 740 745 750 Leu Pro Pro Phe Gly Glu Leu Pro Cys Leu Glu Ser Leu Glu Leu Gln 755 760 765 Asp Gly Ser Val Glu Val Glu Tyr Val Glu Asp Ser Gly Phe Leu Thr 770 775 780 Arg Arg Arg Phe Pro Ser Leu Arg Lys Leu His Ile Gly Gly Phe Cys 785 790 795 800 Asn Leu Lys Gly Leu Gln Arg Met Lys Gly Ala Glu Gln Phe Pro Val 805 810 815 Leu Glu Glu Met Lys Ile Ser Asp Cys Pro Met Phe Val Phe Pro Thr 820 825 830 Leu Ser Ser Val Lys Lys Leu Glu Ile Trp Gly Glu Ala Asp Ala Gly 835 840 845 Gly Leu Ser Ser Ile Ser Asn Leu Ser Thr Leu Thr Ser Leu Lys Ile 850 855 860 Phe Ser Asn His Thr Val Thr Ser Leu Leu Glu Glu Met Phe Lys Asn 865 870 875 880 Leu Glu Asn Leu Ile Tyr Leu Ser Val Ser Phe Leu Glu Asn Leu Lys 885 890 895 Glu Leu Pro Thr Ser Leu Ala Ser Leu Asn Asn Leu Lys Cys Leu Asp 900 905 910 Ile Arg Tyr Cys Tyr Ala Leu Glu Ser Leu Pro Glu Glu Gly Leu Glu 915 920 925 Gly Leu Ser Ser Leu Thr Glu Leu Phe Val Glu His Cys Asn Met Leu 930 935 940 Lys Cys Leu Pro Glu Gly Leu Gln His Leu Thr Thr Leu Thr Ser Leu 945 950 955 960 Lys Ile Arg Gly Cys Pro Gln Leu Ile Lys Arg Cys 965 970 62 945 PRT Artificial Sequence Description of Artificial Sequence alignment SH20-tub 62 Met Ala Glu Ala Phe Ile Gln Val Leu Leu Glu Asn Ile Thr Ser Phe 1 5 10 15 Ile Gln Gly Glu Leu Gly Leu Leu Leu Gly Phe Glu Asn Asp Phe Glu 20 25 30 Asn Ile Ser Ser Arg Phe Ser Thr Ile Gln Ala Val Leu Glu Asp Ala 35 40 45 Gln Glu Lys Gln Leu Lys Asp Lys Ala Ile Lys Asn Trp Leu Gln Lys 50 55 60 Leu Asn Ala Ala Val Tyr Lys Val Asp Asp Leu Leu Asp Glu Cys Lys 65 70 75 80 Ala Ala Arg Leu Glu Gln Ser Arg Leu Gly Cys His His Pro Lys Ala 85 90 95 Ile Val Phe Arg His Lys Ile Gly Lys Arg Ile Lys Glu Met Met Glu 100 105 110 Lys Leu Asp Ala Ile Ala Lys Glu Arg Thr Asp Phe His Leu His Glu 115 120 125 Lys Ile Ile Glu Arg Gln Val Ala Arg Pro Glu Thr Gly Phe Val Leu 130 135 140 Thr Glu Pro Gln Val Tyr Gly Arg Asp Lys Glu Glu Asp Glu Ile Val 145 150 155 160 Lys Ile Leu Ile Asn Asn Val Ser Asn Ala Gln Glu Leu Ser Val Leu 165 170 175 Pro Ile Leu Gly Met Gly Gly Leu Gly Lys Thr Thr Leu Ala Gln Met 180 185 190 Val Phe Asn Asp Gln Arg Val Thr Glu His Phe Tyr Pro Lys Ile Trp 195 200 205 Ile Cys Val Ser Asp Asp Phe Asp Glu Lys Arg Leu Ile Glu Asn Ile 210 215 220 Ile Gly Asn Ile Glu Arg Ser Ser Leu Asp Val Lys Asp Leu Ala Ser 225 230 235 240 Phe Gln Lys Lys Leu Gln Gln Leu Leu Asn Gly Lys Arg Tyr Leu Leu 245 250 255 Val Leu Asp Asp Val Trp Asn Glu Asp Gln Gln Lys Trp Asp Asn Leu 260 265 270 Arg Val Val Leu Lys Val Gly Ala Ser Gly Ala Ser Val Leu Thr Thr 275 280 285 Thr Arg Leu Glu Lys Val Gly Ser Ile Met Gly Thr Leu Gln Pro Tyr 290 295 300 Gln Leu Ser Asn Leu Ser Gln Asp Asp Cys Trp Leu Leu Phe Ile Gln 305 310 315 320 Arg Ala Phe Arg His Gln Glu Glu Ile Ser Pro Asn Leu Val Ala Ile 325 330 335 Gly Lys Glu Ile Val Lys Lys Ser Gly Gly Val Pro Leu Ala Ala Lys 340 345 350 Thr Leu Gly Gly Leu Leu Arg Phe Lys Arg Glu Lys Arg Glu Trp Glu 355 360 365 His Val Arg Asp Ser Glu Ile Trp Asn Leu Pro Gln Asp Glu Met Ser 370 375 380 Ile Leu Pro Ala Leu Arg Leu Ser Tyr His His Leu Pro Leu Ala Leu 385 390 395 400 Arg Gln Cys Phe Ala Tyr Cys Ala Val Phe Pro Lys Asp Thr Lys Met 405 410 415 Glu Lys Lys Lys Val Ile Ser Leu Trp Met Ala His Gly Phe Leu Leu 420 425 430 Ser Arg Arg Asn Leu Glu Leu Glu Asp Val Arg Asn Glu Gly Trp Asn 435 440 445 Glu Leu Tyr Leu Arg Ser Phe Phe Gln Glu Ile Glu Val Arg Tyr Gly 450 455 460 Asn Thr Tyr Phe Lys Met His Asp Leu Ile His Asp Leu Ala Thr Ser 465 470 475 480 Leu Phe Ser Ala Asn Thr Ser Ser Ser Asn Ile Arg Glu Ile Asn Val 485 490 495 Glu Ser Tyr Thr His Met Met Ser Ile Gly Phe Ser Glu Val Val Ser 500 505 510 Ser Tyr Ser Pro Ser Leu Leu Gln Lys Phe Val Ser Leu Arg Val Leu 515 520 525 Asn Leu Ser Tyr Ser Lys Phe Glu Glu Leu Pro Ser Ser Ile Gly Asp 530 535 540 Leu Val His Leu Arg Tyr Met Asp Leu Ser Asn Asn Ile Glu Ile Arg 545 550 555 560 Ser Leu Pro Lys Gln Leu Cys Lys Leu Gln Asn Leu Gln Thr Leu Asp 565 570 575 Leu Gln Tyr Cys Thr Arg Leu Cys Cys Leu Pro Lys Gln Thr Ser Lys 580 585 590 Leu Gly Ser Leu Arg Asn Leu Leu Leu His Gly Cys His Arg Leu Thr 595 600 605 Arg Thr Pro Pro Arg Ile Gly Ser Leu Thr Cys Leu Lys Thr Leu Gly 610 615 620 Gln Ser Val Val Lys Arg Lys Lys Gly Tyr Gln Leu Gly Glu Leu Gly 625 630 635 640 Ser Leu Asn Leu Tyr Gly Ser Ile Lys Ile Ser His Leu Glu Arg Val 645 650 655 Lys Asn Asp Lys Glu Ala Lys Glu Ala Asn Leu Ser Ala Lys Glu Asn 660 665 670 Leu His Ser Leu Ser Met Lys Trp Asp Asp Asp Glu Pro His Arg Tyr 675 680 685 Glu Ser Glu Glu Val Glu Val Leu Glu Ala Leu Lys Pro His Ser Asn 690 695 700 Leu Thr Cys Leu Lys Ile Ser Gly Phe Arg Gly Ile Arg Leu Pro Asp 705 710 715 720 Trp Met Asn His Ser Val Leu Lys Asn Ile Val Leu Ile Glu Ile Ser 725 730 735 Gly Cys Lys Asn Cys Ser Cys Leu Pro Pro Phe Gly Asp Leu Pro Cys 740 745 750 Leu Glu Ser Leu Glu Leu Tyr Arg Gly Ser Ala Glu Tyr Val Glu Glu 755 760 765 Val Asp Ile Asp Val Asp Ser Gly Phe Pro Thr Arg Ile Arg Leu Pro 770 775 780 Ser Leu Arg Lys Leu Cys Ile Cys Lys Phe Asp Asn Leu Lys Gly Leu 785 790 795 800 Leu Lys Lys Glu Gly Gly Glu Gln Phe Pro Val Leu Glu Glu Met Glu 805 810 815 Ile Arg Tyr Cys Pro Ile Pro Thr Leu Ser Pro Asn Leu Lys Ala Leu 820 825 830 Thr Ser Leu Asn Ile Ser Asp Asn Lys Glu Ala Thr Ser Phe Pro Glu 835 840 845 Glu Met Phe Lys Ser Leu Ala Asn Leu Lys Tyr Leu Asn Ile Ser His 850 855 860 Phe Lys Asn Leu Lys Glu Leu Pro Thr Ser Leu Ala Ser Leu Asn Ala 865 870 875 880 Leu Lys Ser Leu Lys Ile Gln Trp Cys Cys Ala Leu Glu Asn Ile Pro 885 890 895 Lys Glu Gly Val Lys Gly Leu Thr Ser Leu Thr Glu Leu Ile Val Lys 900 905 910 Phe Ser Lys Val Leu Lys Cys Leu Pro Glu Gly Leu His His Leu Thr 915 920 925 Ala Leu Thr Arg Leu Lys Ile Trp Gly Cys Pro Gln Leu Ile Lys Arg 930 935 940 Cys 945 63 945 PRT Artificial Sequence Description of Artificial Sequence alignment T118-tar 63 Met Ala Glu Ala Phe Ile Gln Val Leu Leu Glu Asn Ile Thr Ser Phe 1 5 10 15 Ile Gln Gly Glu Leu Gly Leu Leu Leu Gly Phe Glu Asn Glu Phe Glu 20 25 30 Asn Ile Ser Ser Arg Phe Ser Thr Ile Gln Ala Val Leu Glu Asp Ala 35 40 45 Gln Glu Lys Gln Leu Lys Asp Lys Ala Ile Lys Asn Trp Leu Gln Lys 50 55 60 Leu Asn Ala Ala Ala Tyr Lys Val Asp Asp Leu Leu Asp Glu Cys Lys 65 70 75 80 Ala Ala Arg Leu Glu Gln Ser Arg Leu Gly Arg His His Pro Lys Ala 85 90 95 Ile Val Phe Arg His Lys Ile Gly Lys Arg Ile Lys Glu Met Met Glu 100 105 110 Lys Leu Asp Ala Ile Ala Lys Glu Arg Thr Asp Phe His Leu His Glu 115 120 125 Lys Ile Ile Glu Arg Gln Val Ala Arg Pro Glu Thr Gly Pro Val Leu 130 135 140 Thr Glu Pro Gln Val Tyr Gly Arg Asp Lys Glu Glu Asp Glu Ile Val 145 150 155 160 Lys Ile Leu Ile Asn Asn Val Ser Asn Ala Leu Glu Leu Ser Val Leu 165 170 175 Pro Ile Leu Gly Met Gly Gly Leu Gly Lys Thr Thr Leu Ala Gln Met 180 185 190 Val Phe Asn Asp Gln Arg Val Thr Glu His Phe Tyr Pro Lys Ile Trp 195 200 205 Ile Cys Val Ser Asp Asp Phe Asp Glu Lys Arg Leu Ile Glu Thr Ile 210 215 220 Ile Gly Asn Ile Glu Arg Ser Ser Leu Asp Val Lys Asp Leu Ala Ser 225 230 235 240 Phe Gln Lys Lys Leu Gln Gln Leu Leu Asn Gly Lys Arg Tyr Leu Leu 245 250 255 Val Leu Asp Asp Val Trp Asn Glu Asp Gln Gln Lys Trp Asp Asn Leu 260 265 270 Arg Ala Val Leu Lys Val Gly Ala Ser Gly Ala Ser Val Leu Thr Thr 275 280 285 Thr Arg Leu Glu Lys Val Gly Ser Ile Met Gly Thr Leu Gln Pro Tyr 290 295 300 Gln Leu Ser Asn Leu Ser Gln Asp Asp Cys Trp Leu Leu Phe Ile Gln 305 310 315 320 Arg Ala Tyr Arg His Gln Glu Glu Ile Ser Pro Asn Leu Val Ala Ile 325 330 335 Gly Lys Glu Ile Val Lys Lys Ser Gly Gly Val Pro Leu Ala Ala Lys 340 345 350 Thr Leu Gly Gly Leu Leu Arg Phe Lys Arg Glu Lys Arg Glu Trp Glu 355 360 365 His Val Arg Asp Ser Glu Ile Trp Asn Leu Pro Gln Asp Glu Met Ser 370 375 380 Ile Leu Pro Val Leu Arg Leu Ser Tyr His His Leu Pro Leu Asp Leu 385 390 395 400 Arg Gln Cys Phe Ala Tyr Cys Ala Val Phe Pro Lys Asp Thr Lys Met 405 410 415 Glu Lys Lys Lys Val Ile Ser Leu Trp Met Ala His Gly Phe Leu Leu 420 425 430 Ser Arg Arg Asn Leu Glu Leu Glu Asp Val Gly Asn Glu Val Trp Asn 435 440 445 Glu Leu Tyr Leu Arg Ser Phe Phe Gln Glu Ile Glu Val Arg Tyr Gly 450 455 460 Asn Thr Tyr Phe Lys Met His Asp Leu Ile His Asp Leu Ala Thr Ser 465 470 475 480 Leu Phe Ser Ala Asn Thr Ser Ser Ser Asn Ile Arg Glu Ile Asn Val 485 490 495 Glu Ser Tyr Thr His Met Met Ser Ile Gly Phe Ser Glu Val Val Ser 500 505 510 Ser Tyr Ser Pro Ser Leu Leu Gln Lys Phe Val Ser Leu Arg Val Leu 515 520 525 Asn Leu Ser Tyr Ser Lys Phe Glu Glu Leu Pro Ser Ser Ile Gly Asp 530 535 540 Leu Val His Leu Arg Tyr Met Asp Leu Ser Asn Asn Ile Glu Ile Arg 545 550 555 560 Ser Leu Pro Lys Gln Leu Cys Lys Leu Gln Asn Leu Gln Thr Leu Asp 565 570 575 Leu Gln Tyr Cys Thr Arg Leu Cys Cys Leu Pro Lys Gln Thr Ser Lys 580 585 590 Leu Gly Ser Leu Arg Asn Leu Leu Leu His Gly Cys His Arg Leu Thr 595 600 605 Arg Thr Pro Pro Arg Ile Gly Ser Leu Thr Cys Leu Lys Thr Leu Gly 610 615 620 Gln Phe Val Val Gly Arg Lys Lys Gly Tyr Gln Leu Gly Glu Leu Gly 625 630 635 640 Ser Leu Asn Leu Tyr Gly Ser Ile Lys Ile Ser His Leu Glu Arg Val 645 650 655 Lys Asn Asp Lys Glu Ala Lys Glu Ala Asn Leu Ser Ala Lys Glu Asn 660 665 670 Leu His Ser Leu Ser Met Lys Trp Asp Asp Asp Glu Pro His Arg Tyr 675 680 685 Glu Ser Glu Glu Val Glu Val Leu Glu Ala Leu Lys Pro His Ser Asn 690 695 700 Leu Thr Cys Leu Thr Ile Ser Gly Phe Arg Gly Ile Arg Leu Pro Asp 705 710 715 720 Trp Met Asn His Ser Val Leu Lys Asn Ile Val Leu Ile Glu Ile Ser 725 730 735 Gly Cys Lys Asn Cys Ser Cys Leu Pro Pro Phe Gly Asp Leu Pro Cys 740 745 750 Leu Glu Ser Leu Gln Leu Tyr Arg Gly Ser Ala Glu Tyr Val Glu Glu 755 760 765 Val Asp Ile Asp Val Asp Ser Gly Phe Pro Thr Arg Ile Arg Phe Pro 770 775 780 Ser Leu Arg Lys Leu Cys Ile Cys Lys Phe Asp Asn Leu Lys Gly Leu 785 790 795 800 Val Lys Lys Glu Gly Gly Glu Gln Phe Pro Val Leu Glu Glu Met Glu 805 810 815 Ile Arg Tyr Cys Pro Ile Pro Thr Leu Ser Ser Asn Leu Lys Ala Leu 820 825 830 Thr Ser Leu Asn Ile Ser Asp Asn Lys Glu Ala Thr Ser Phe Pro Glu 835 840 845 Glu Met Phe Lys Ser Leu Ala Asn Leu Lys Tyr Leu Asn Ile Ser His 850 855 860 Phe Lys Asn Leu Lys Glu Leu Pro Thr Ser Leu Ala Ser Leu Asn Ala 865 870 875 880 Leu Lys Ser Leu Lys Ile Gln Trp Cys Cys Ala Leu Glu Ser Ile Pro 885 890 895 Glu Glu Gly Val Lys Gly Leu Thr Ser Leu Thr Glu Leu Ile Val Lys 900 905 910 Phe Cys Lys Met Leu Lys Cys Leu Pro Glu Gly Leu Gln His Leu Thr 915 920 925 Ala Leu Thr Arg Val Lys Ile Trp Gly Cys Pro Gln Leu Ile Lys Arg 930 935 940 Cys 945 

1. An isolated or recombinant nucleic acid comprising a nucleic acid coding for the amino acid sequence of FIG. 8 or a functional fragment or a homologue thereof.
 2. A fragment according to claim 1 coding for the leucine rich repeat (LRR) fragment of the amino acid sequence of FIG.
 8. 3. A nucleic acid according to claim 1 or 2, said nucleic acid encoding a gene product that is capable of providing a member of the Solanaceae family with resistance against an oomycete pathogen, or a functional equivalent thereof.
 4. A nucleic acid according to claim 3 wherein said member of the Solanaceae family comprises S. tuberosum.
 5. A nucleic acid according to claim 3 where said resistance is race non-specific.
 6. A nucleic acid according to claim 1 to 5 comprising a sequence as depicted in FIG. 6 for Rpi-blb or part thereof.
 7. A nucleic acid according to claim 1 to 5 at least comprising a LRR domain.
 8. A vector comprising a nucleic acid according to anyone of claims 1 to
 7. 9. A host cell comprising a nucleic acid according to anyone of claims 1 to 7 or a vector according to claim
 8. 10. A cell according to claim 9 comprising a plant cell.
 11. A cell according to claim 10 wherein said plant comprises a member of the Solanaceae family.
 12. A plant comprising a cell according to anyone of claims 9 to
 11. 13. A part derived from a plant according to claim
 12. 14. A part according to claim 13 wherein said tuber comprises a potato or said fruit comprises a tomato.
 15. Progeny of a plant according to claim
 12. 16. A proteinaceous substance encoded by a nucleic acid according to anyone of claims 1 to
 7. 17. A proteinaceous substance comprising an amino acid sequence as depicted in FIG. 8 or a functional equivalent thereof.
 18. A binding molecule directed at a nucleic acid according to anyone of claim 1 to
 7. 19. A binding molecule according to claim 18 comprising a probe or primer.
 20. A binding molecule according to claim 18 or 19 provided with a label.
 21. A binding molecule according to claim 20 wherein said label comprises an excitable moiety.
 22. Use of a nucleic acid according to anyone of claims 1 to 7 or a vector according to claim 8 or a cell according to anyone of claims 9 to 11 or a substance according to claim 16 or 17 or a binding molecule according to anyone of claims 18 to 21 in a method for providing a plant or its progeny with resistance against an oomycete infection.
 23. Use according to claim 22 wherein said oomycete comprises Phytophthora infestans.
 24. Use according to claim 22 or 23 wherein said plant comprises S. tuberosum.
 25. A method for providing a plant or its progeny with at least partial resistance against an oomycete infection comprising providing said plant or part thereof with a gene or functional fragment thereof comprising a nucleic acid corresponding to one of a cluster of genes identifiable by phylogenetic tree analyses as corresponding to the Rpi-blb, RCG1-blb, RCG3-blb and RCG4-blb cluster of FIG. 9, said nucleic acid encoding a gene product that is capable of providing a member of the Solanaceae with resistance against an oomycete fungus, or providing said plant or part thereof with a nucleic acid according to anyone of claims 1 to 7 or a vector according to claim 8 or a cell according to claims 9-11 or a substance according to claim 16 or
 17. 26. A method for selecting a plant or plant material or progeny thereof for its susceptibility or resistance to an oomycete infection comprising testing at least part of said plant or plant material or progeny thereof for the presence or absence of a nucleic acid corresponding to one of a cluster of genes identifiable by phylogenetic tree analyses as corresponding to the Rpi-blb, RCG1-blb, RCG3-blb and RCG4-blbcluster of FIG. 9, said nucleic acid encoding a gene product that is capable of providing a member of the Solanaceae with resistance against an oomycete fungus.
 27. A method according to claim 26 comprising contacting at least part of said plant or plant material or progeny thereof with a binding molecule according to anyone of claims 18 to 21 and determining the binding of said molecule to said part.
 28. A method according to claim 27 wherein said oomycete comprises Phytophthora infestans.
 29. A method according to claim 27 or 28 wherein said plant comprises S. tuberosum.
 30. An isolated S. bulbocastanum, or part thereof, susceptible to an oomycete infection caused by Phytophthora infestans. 